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Programme Book

29 October - 01 November 2017Berlin, Germany

Scientific Programme CommitteeJessica Downs (Chair) || Susan Gasser || Penny Jeggo

Protecting the Code Epigenetic Impacts on Genome Stability

25th Biennial Congress of the European Association for Cancer ResearchFrom Fundamental Insight to Rational Cancer Treatment

EACR 2530 June - 03 July 2018 Amsterdam

SAVE THE DATEfor this landmark congress celebrating 50 years of the EACR

051968 - 2018YEARS

www.eacr25.org #EACR25

Protecting the Code: Epigenetic Impacts on Genome StabilityBerlin, Germany, 29 October - 01 November 2017

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29 October - 01 November 2017 • Berlin, Germany

Protecting the CodeEpigenetic Impacts on Genome Stability

Day 1 - Sunday 29 October

15.30 – 17.00 REGISTRATION Planck Lobby

17.00 – 17.15 WELCOME & INTRODUCTION Scientific Programme Committee

17.15 – 18.00 Keynote Lecture 1 Q&A: 18.00 – 18.15 Akira Yasui Tohoku University, Japan “Histone modification and nucleosome remodeling regulating transcription and DNA repair”

18.15 – 19.00 SPEED NETWORKING Planck Lobby

19.00 – 20.30 WELCOME BUFFET Meitner Hall Trade exhibition opens

Day 2 - Monday 30 October

08.30 – 09.00 POSTER PREVIEW Meitner Hall Coffee available

SESSION 1: CHROMATIN REMODELERS AND DNA DAMAGE Session Chair: Susan Gasser

09.00 – 09.20 Claudia Lukas NNF CPR, Denmark Q&A: 09.20 – 09.30 “The role of DNA replication stress in cell fate decisions after whole-genome duplication”

09.30 – 09.50 Niels Mailand NNF CPR, DenmarkQ&A: 09.50 – 10.00 “Regulation of chromatin ubiquitylation in the DNA damage response”


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10.00 – 10.10 Aaron Goodarzi University of Calgary, CanadaQ&A: 10.10 – 10.15 Proffered Paper 1: ”The CHD6 chromatin remodeler is an oxidative stress-induced DNA damage response factor”

10.15 – 10.25 EXHIBITOR INTRODUCTIONS

10.25 – 11.00 COFFEE BREAK

11.00 – 11.10 Boris Pfander Max Planck Institute of Biochemistry, Germany Q&A: 11.10 – 11.15 Proffered Paper 2: “Chromatin constitutes a bottleneck in the response to double strand breaks”

11.15 – 11.35 Jessica Downs ICR, UKQ&A: 11.35 – 11.45 “The PBAF chromatin remodelling complex and maintenance of genome stability”

11.45 – 12.15 SATELLITE SYMPOSIUM: CHROMATRAP

12.15 – 13.00 LUNCH Restaurant

13.00 – 14.30 POSTER DEFENCE SESSION 1 & TRADE EXHIBITION Meitner Hall Odd numbered posters (1, 3, 5, etc.) will be presented

SESSION 2: PROPAGATING THE EPIGENOME Session Chair: Jo Morris

14.30 – 14.50 Anja Groth BRIC, DenmarkQ&A: 14.50 – 15.00 “Chromatin replication and epigenome maintenance”

15.00 – 15.20 Iestyn Whitehouse MSKCC, USA Q&A: 15.20 – 15.30 “Coupling of genes and replication origins”

15.30 – 15.40 Kyle Miller University of Texas at Austin, USAQ&A: 15.40 – 15.45 Proffered Paper 3: “Bromodomain proteins: Readers of Genome Integrity”

15.45 – 16.05 Kristijan Ramadan University of Oxford, UKQ&A: 16.05 – 16.15 “Ubiqutin dependent ATPase p97 and deubiquitinating enzyme X (DUBX) regulate DNA damage response after ionising radiation”

16.15 – 17.30 ROUND TABLE DISCUSSIONS Einstein Lounge Beverages are available to purchase from the bar.

17.30 FREE EVENING TO EXPLORE BERLIN


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Day 3 - Tuesday 31 October

08.30 – 09.00 POSTER PREVIEW Coffee available

09.00 – 09.30 MEET THE EXPERT Penny Jeggo University of Sussex, UK

SESSION 3: DNA DAMAGE REPAIR Session Chair: Iestyn Whitehouse

09.30 – 09.50 Haico van Attikum LUMC, NetherlandsQ&A: 09.50 – 10.00 “Regulation and dynamics of DNA repair in a chromatin context”

10.00 – 10.10 Jacques Cote Université Laval, CanadaQ&A: 10.10 – 10.15 Proffered Paper 4: “Phospho-dependent recruitment of NuA4 by MRX at DNA breaks regulates RPA dynamics during resection”

10.15 – 10.25 Ulrich Rass Friedrich Miescher Institute, SwitzerlandQ&A: 10.25 – 10.30 Proffered Paper 5: “Helicases and nucleases determining replication fork recovery or demise, and implications for genome stability and human disease”


11.00 – 11.20 Brendan Price Dana-Farber Cancer Institute, USAQ&A: 11.20 – 11.30 “Constructing specialized, acetylated chromatin domains to promote DSB repair”

11.30 – 11.50 Jo Morris University of Birmingham, UKQ&A: 11.50 – 12.00 “The SUMO bomb in DNA double-strand break repair”

12.00 – 12.10 Amélie Fradet-Turcotte Université Laval, CanadaQ&A: 12.10 – 12.15 Proffered Paper 6: “A new class of ubiquitylated-histone readers involved in the response to DNA damage”

12.15 – 13.00 LUNCH Restaurant

13.00 – 14.30 POSTER DEFENCE SESSION 2 & TRADE EXHIBITION Meitner Hall Even numbered posters (2, 4, 6, etc.) will be presented


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SESSION 4: CHROMATIN AND DISEASE STATES Session Chair: Penny Jeggo

14.30 – 14.50 Tim Humphrey University of Oxford, UKQ&A: 14.50 – 15.00 “Histone H3K36 trimethylation, genome stability and cancer”

15.00 – 15.10 Ashby Morrison Stanford University, USAQ&A: 15.10– 15.15 Proffered Paper 7: “Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis”

15.15 – 15.35 Fabrizio d’Adda di Fagagna IFOM, ItalyQ&A: 15.35 – 15.45 “DNA damage response activation in cancer and the role of non coding RNAs”

15.45 – 16.30 Keynote Lecture 2Q&A: 16.30 – 16.45 Geneviève Almouzni Institute Curie, France “Shaping chromatin in the nucleus, the bricks and the architects”

19.30 DRINKS RECEPTION & CONFERENCE DINNER Pre-booked optional extra

Day 4 - Wednesday 01 November

SESSION 5: CHROMATIN REMODELERS AT THE TRANSCRIPTION/REPLICATION INTERFACE Session Chair: Jessica Downs

09.00 – 09.20 Manolis Papamichos-Chronakis Newcastle University, UKQ&A: 09.20 – 09.30 “INO80 chromatin remodeller links RNA quality control to transcriptional regulation”

09.30 – 09.50 Philipp Oberdoerffer National Cancer Institute, USAQ&A: 09.50 – 10.00 “Replication stress in chromatin: getting fragile sites in shape”

10.00 – 10.10 Andrew Wood IGMM Edinburgh, UKQ&A: 10.10 – 10.15 Proffered Paper 8: “Imprinted genes as a model system to study the impact of chromatin on targeted mutagenesis”



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10.45 – 10.55 Sophie Polo Epigenetics and Cell Fate, France Q&A: 10.55 – 11.00 Proffered Paper 9: “Epigenome maintenance in response to DNA damage”

11.00 – 11.20 Gaëlle Legube CBI Toulouse, FranceQ&A: 11.20 – 11.30 “Chromatin and chromosome dynamics following DNA Double Strand Break”

11.30 – 12.00 The EMBO Keynote LectureQ&A: 12.00 – 12.15 Susan Gasser FMI, Switzerland “Remodelers in response to DNA damage”

12.15 – 12.30 CLOSING REMARKS AND PRESENTATION OF AWARDS Scientific Programme Committee

12.30 LUNCH & DEPART A packed lunch will be provided

Congratulations to the winners of the EACR-Worldwide Cancer Research Meeting Bursaries. Each winner received a full registration free of charge and funds of up to 500 Euros to assist with the cost of travel.

EACR-Worldwide Cancer Research Meeting Bursary Award Winners

Marta Baldascini Ireland

Sandra Segura-Bayona Spain

Marwa Tantawy Egypt


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Molecular Pathology Approach to Cancer

8th EACR-OECI Joint Course 04 - 06 June 2018AMSTERDAM NETHERLANDS

Save the Date...

For more information visit www.biologists.com

The Company of Biologists is a not-for-profit publishing organisation dedicated to supporting and inspiring the biological community. We are run by distinguished practicing scientists. We exist to profit science, not shareholders. We inspire new thinking and support the worldwide community of biologists.

We do this by publishing leading peer-reviewed journals, facilitating scientific meetings and communities, providing travel grants for young researchers and by supporting societies and facilitating communities.

Grants and FellowshipsWe use the surplus we generate for the benefit of biology, supporting and encouraging the sharing of knowledge across the biological community by funding various Grants and Travelling Fellowships.

Visit www.biologists.com/grants or www.biologists.com/travelling-fellowships

Workshops and Meetings Through our regular Workshops and Journal Meetings, we help establish and develop professional networks that disseminate knowledge and strengthen personal connections across the scientific community.

Visit www.biologists.com/workshops orwww.biologists.com/meetings


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The organisers wish to express their appreciation for the significant support provided by sponsors at the EACR Conference Protecting the Code: Epigenetic Impacts on Genome Stability. Their interest and enthusiasm for the conference has enabled the organisers to provide an impressive scientific programme.

Elite Sponsor

Exhibitors

Partners and Sponsors

Grants


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Monday 30 October 2017, 11.45 – 12.15

Sahar Osman & Lindsay Parkes

“A more efficient, sensitive and robust method of chromatin immunoprecipitation (ChIP)”

Satellite Symposium - Elite Sponsor

We are pleased to announce that Chromatrap will not only be exhibiting at the conference but also inviting participants to join a Satellite Symposium.

EACR Sustaining Members

The European Association for Cancer Research gratefully acknowledges the organisations that support the Association as Sustaining Members. Sustaining Members offer ongoing support to the EACR and provide the means for the Association to develop important initiatives. The EACR Conference Series is an example of this.

Chromatrap will be presenting the company, its novel solid state technology and its application in epigenetic research. We will introduce our product range highlighting our popular chromatin immunoprecipitation kits and how they can advance routine experiments. Final talking points will include ongoing projects, product developments and their repertoire of guides and resources.


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Meet the Exhibitors

Abcam Website: www.abcam.comContact: [email protected] at the conference by: Davide Mantiero, Christian Lautenschlager

Describe Abcam in 5 words or lessAccelerating scientific discovery

Tell us a little bit about Abcam As an innovator in reagents and tools, Abcam serves life science researchers globally to achieve their mission, faster. Providing the research and clinical communities with tools and scientific support, Abcam offers highly validated binders and assays to address important targets in critical biological areas such as epigenetics.

Why are you attending the conference? Who would you like to meet at the conference?We are keen to learn what new and exciting advances scientists have made in epigenetics and genome stability research, and meet with researchers from this field to discuss how we can help them accelerating their research.

Active Motif Website: www.activemotif.com Contact: [email protected], [email protected] Represented at the conference by: Agnieszka Siekaniec, Sarantis Chlamydas

Describe Active Motif in 5 words or lessActive Motif – enabling epigenetic research

Tell us a little bit about Active Motif Active Motif is developing and delivering innovative tools to enable epigenetics and gene regulation research. Active Motif offers a comprehensive portfolio of epigenetics-related products and services and the support of our team of epigenetic experts to provide complete and innovative solutions to tackle your scientific inquiries.

Why are you attending the conference? Who would you like to meet at the conference?Conferences provide excellent opportunities to expand the knowledge about new discoveries and help the scientists improve their research by discussion their findings with a greater audience. A big reason for me to going to conferences is to meet new people from the field of Epigenetics, discuss with them and creates an opportunity to build partnerships.


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BioCat and EpiGentek Website: www.biocat.comContact: [email protected] or +49 6221 7141516Represented at the conference by: Dr. Kambiz Navabi

Describe BioCat in 5 words or lessInnovative products for life-science research

Tell us a little bit about BioCat Through our long-lasting partnership with Epigentek, a leading developer and provider of innovative technologies and products for epigenetic-related research, BioCat provides kits and services facilitating the analysis of epigenetic regulation in academic research labs as well as in pharma & biotech industry in Austria, Germany and Switzerland.

Why are you attending the conference? Who would you like to meet at the conference?BioCat attends the conference to meet existing and potential new customers and to present unique products for epigenetics research that are specifically designed to make assays much simpler, faster, more convenient, and highly more efficient than currently used methods.

ChromatrapWebsite: www.chromatrap.comContact: [email protected] Represented at the conference by: Lindsay Parkes and Sahar Osman

Describe Chromatrap in 5 words or lessInnovative, simple, reliable, bold, efficient

Tell us a little bit about ChromatrapChromatrap is a life science company enabling the advancement of epigenetic research through its revolutionary solid-state system. The novel range of ChIP kits eliminates the laborious use of beads allowing researchers to perform faster, easier and more sensitive experiments for epigenetics research.

Why are you attending the conference? Who would you like to meet at the conference?Chromatrap is here to shake up the world of epigenetics techniques by making its solid-state based kits the standard way of performing ChIP. We would love to meet academics to understand how Chromatrap can help their research and industry leaders to continue to innovate.

The conference will give plenty of opportunities for you to meet representatives from companies working in the same field.

To speak to our exhibitors and find out more about them, please visit the stands in the trade exhibition.


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Interactive activities at the ConferenceAn important part of the EACR Conference Series is the range of opportunities we aim to provide for participants to interact, discuss, reflect and build relationships and collaborations.

We hope you enjoy the dedicated interactive activities, which are listed below.

Speed Networking18.15 – 19.00 Sunday 29 October 2017

The first evening will begin with this informal (and hopefully fun!) session, giving all participants the opportunity to make lots of new connections in a short space of time. Use the starter questions on the tall tables, and find a different table when you hear the bell ring.

Poster Defence Sessions

13.00 – 14.30 Monday 30 October 13.00 – 14.30 Tuesday 31 October

There are two dedicated Poster Defence Sessions in the programme. At these times, the presenters for that session are asked to stand by their posters to discuss their work with other participants and invited speakers.

Two EACR Poster Prizes worth €100 each will be awarded to the best poster presentations at the conference. The judging panel is comprised of speakers from the conference, and they will assess the top scoring abstracts based on the scientific content, the layout of the poster, and the verbal discussion. The winners will be announced during the Closing Summary on Wednesday.

Poster Preview

08.30 – 09.00 Monday 30 October 08.30 – 09.00 Tuesday 31 October

Monday and Tuesday will begin with an optional poster viewing slot. Participants are invited to use this time for further discussion in the poster areas, but presenters are not required to be by their posters at these sessions. Coffee and tea will be offered during these times.

Round Table Discussions

16.15 – 17.30 Monday 30 October

This session provides the opportunity for participants to engage with invited speakers in small groups, to ask questions and converse informally. The bar will be


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open for the opportunity to purchase drinks.

Meet the Expert

09.00 – 09.30 Tuesday 31 October

Professor Penny Jeggo has made important contributions to our understanding of DNA damage response and human radiosensitivity. Penny will discuss how science has changed during her career, the challenges that come with running a lab, and what it takes to be successful in science. There will be time for questions and discussion.

Conference Dinner

19.30 – 22.00 Tuesday 31 October 2017

The Conference Dinner will take place on the evening of Tuesday 31 October 2017, the final night of the conference. It will be an excellent opportunity for participants and speakers to get to know each other in a relaxed and informal environment. The evening will begin with a Drinks Reception in the Planck Lobby of Harnack House at 19.30, featuring live music. The three-course served dinner will take place downstairs in the atmospheric Liebig Vault from 20.00. Tickets must be purchased in advance.

Don’t forget to let us have your feedback about these activities in the survey we will send after the conference!

Conference Dinner at Harnack House.

worldwidecancerresearch.org

Worldwide Cancer Research is a charity registered in Scotland, No: SC022918

From the world’s best research institutions and renowned specialists to unexpected and diverse projects by up and coming talent. We fund all types of research and for one very good reason – to gain a global perspective. Because research doesn’t happen in isolation. And the answers will not come from one scientist, in one lab, in one country. That’s why Worldwide Cancer Research are prepared for whatever it takes and wherever it takes us.

We fund research into any type of cancer anywhere in the world.


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Speaker abstractsHistone modification and nucleosome remodeling regulating transcription and DNA repair

Akira Yasui11 Institute of Development, Aging and Cancer (IDAC)/Tohoku University, Sendai, JAPAN

Histone modifications play important roles in DNA damage response and DNA repair. Mono-ubiquitination of histone H2A is a transcriptionally repressive mark with reversible nature. We recently reported that a transcriptional elongation factor, ENL in RNA PolII complex is phosphorylated at its conserved SQ sites by ATM activated by DSB. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs and ubiquitinates H2A leading to transcriptional repression. ENL and PRC1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Thus, emergency signal of DSB propagated to the sites of RNA PolII complexes by ATM contributes to rapid transcriptional repression alongside the transcription truck and its smooth resumption after repair (1, 2). Another way of chromatin remodeling is nucleosome remodeling, which regulates transcription in an ATP-dependent manner. Suppression of DNA damage-responding BAF factors belonging to SWI/SNF family of nucleosome remodeling makes U2OS cells significantly sensitive to X-rays, UV, and especially to cisplatin and represses the accumulation of repair proteins at DNA damage and DNA repair. Recent cancer genome sequencing and expression analysis have shown that most of the BAF factors are frequently mutated and silenced in various types of cancer cells. While core DNA repair activity is generally stable over age and cell types, the stability of nucleosome remodeling complexes may determine genome integrity in addition to transcriptional regulation, and influence cellular aging (3, 4).

1. Ui A, et al. Mol Cell. 58:468, 2015.

2. Ui A, and Yasui A. Nucleus. 7;138, 2016.

3. Watanabe R, et al. Cancer Res. 74:2465, 2014.

4. Watanabe R,et al. Philos Trans R Soc Lond B Biol Sci. 2017 Oct 5;372.

Speaker abstracts


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Speaker abstracts

The role of DNA replication stress in cell fate decisions after whole-genome duplication

Claudia Lukas1

1 Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DENMARK

It is well established that errors during DNA repair can cause structural and numerical chromosome aberrations and that such genome instability is strongly associated with human diseases such as immune deficiencies, neuropathologies, premature ageing and cancer. Our lab is interested in identifying how human cells protect integrity of their genomes challenged by an endogenous (and thereby inevitable) sources DNA damage that may lead to heritable chromosomal aberrations. Errors during DNA replication are a key contributor to genome instability and cancer, and we have recently uncovered cytokinesis failure at the end of mitosis as a hitherto unrecognized primary source of replication stress. I will present evidence that the inability to separate two otherwise normal nuclei to two daughter cells generates an unusually asymmetric pattern of replication stress, usually in one nucleus, which develops to bona fide DNA damage including DNA double strand breaks. I will then elaborate on whether this specific source of endogenous DNA damage is beneficial in preventing such potentially dangerous cells from further proliferation or whether it rather fuels genome instability in the surviving fraction of cells with acute whole-genome duplication. Furthermore, I will discuss how tetraploidy can be both tumor-promoting and yet essential in several human tissues to execute and maintain differentiation program, with a special emphasis on replication stress as a potential modulator of these cell fate decisions.


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Speaker abstracts

Regulation of chromatin ubiquitylation in the DNA damage response

Niels Mailand1

1 Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DENMARK

To mitigate the threat posed by multitudinous endogenously and exogenously generated genotoxic insults, cells are equipped with a sophisticated DNA damage response (DDR), a global network of coordinated pathways that impact diverse cellular processes to safeguard genome integrity, thereby providing an important cellular barrier towards the onset of severe pathologies such as cancer. Signaling by ubiquitin and ubiquitin-like modifiers orchestrate and regulate cellular responses to DNA damage at multiple levels, often involving extensive crosstalk between these modifications. Non-proteolytic K63-linked polyubiquitylation has a well-established key role in many aspects of the DDR. For instance, we have shown that K63-linked chromatin ubiquitylation mediated by the RNF8-RNF168 E3 ligase cascade is crucial for promoting recruitment of key DNA repair factors to sites of DNA double-strand breaks (DSBs), and that H1-type linker histones are important targets of DSB-induced K63 ubiquitylation in this pathway. How DNA damage-associated K63 ubiquitylation responses are dynamically regulated and terminated is currently less well understood. We are combining systems-wide and focused approaches to illuminate the molecular framework and factors underlying these processes. My presentation at the meeting will focus on a new deubiquitylating enzyme (DUB) involved in regulating K63 ubiquitylation dynamics to promote genome stability.


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Proffered Paper 1

The CHD6 chromatin remodeler is an oxidative stress-induced DNA damage response factor

Shaun Moore2, Fintan KT Stanley2, Martijn Luijsterburg1, Shujuan Fang2, Haico Van Attikum1, Aaron Goodarzi21 Leiden University Medical Centre, Leiden, NETHERLANDS, 2 University of Calgary, Calgary, AB, CANADA

Oxidative stress-induced DNA damage is a threat to the health and survival of a cell. Oxidative DNA damage responses involve nucleosome displacement, exchange or removal by ATP-dependent chromatin remodeling enzymes to promote DNA repair and transcriptional events. Chromatin remodelers adjust nucleosome spacing to regulate DNA accessibility and gene expression in response to stimuli, and are often mutated or abnormally expressed in cancer. We describe a role for the CHD6 (Chromodomain, Helicase, DNA-binding 6) chromatin remodeling enzyme in the response to oxidative stress-induced DNA damage. In response to high O2, H2O2 or ionizing radiation exposure, CHD6 protein levels are stabilized via suppressed proteolytic degradation. CHD6 relocates rapidly to DNA damage caused by microirradiation or KillerRed-induced oxidative stress, but not to enzyme-induced DNA double strand breaks. CHD6 interacts with poly ADP-ribose (PAR), and retention at DNA damage sites is PARP-dependent, prolonged by PARG depletion but is XRCC1-independent. CHD6 ablation by CRISPR causes increased oxidative stress-induced PAR formation per dose of H2O2 relative to controls, while over-expression of CHD6 suppresses PAR-formation. CHD6-deleted cells also display elevated DNA strand breakage, hypersensitive G2/M checkpoint induction, abnormal chromatin relaxation, attenuated anti-oxidant transcriptional responses and decreased survival and cell growth after oxidative stress-induced DNA damage.

Speaker abstracts


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Proffered Paper 2

Chromatin constitutes a bottleneck in the response to double strand breaks

Susanne Bantele1, Claudio Lademann1, Boris Pfander1

1 Max Planck Institute of Biochemistry, Martinsried/Munich, GERMANY

DNA double strand breaks (DSBs) can be repaired by two principal cellular mechanisms – recombination-based (such as homologous recombination, HR) and direct ligation-based (such as non-homologous end joining, NHEJ). In mitotically dividing cells HR critically depends on the presence of sister chromatids as repair templates and hence DSB repair pathway choice is highly cell cycle regulated. Regulation occurs primarily at the level of DNA end resection, the first step in the HR reaction. Notably, nucleosomes restrict resection both directly and indirectly by recruiting nucleosome-associated resection inhibitors such as Rad9/53BP1. Nucleosome remodellers – in particular Fun30/SMARCAD1– are recruited to DSBs in order to overcome this inhibition.

Our data show that budding yeast Fun30 is cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11 and the 9-1-1 complex. This targeting function is required for proper localization of Fun30 to damaged chromatin and efficient long-range resection of DSBs. Remarkably, constitutive targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that nucleosome remodelling during resection is underlying DNA repair pathway choice. Notably, also the human orthologs of yeast Fun30-Dpb11, SMARCAD1-TOPBP1, form a similar CDK-regulated complex, suggesting that CDK-dependent targeting of the chromatin remodeller complex to damaged sites is a conserved mechanism for DSB repair regulation.

Our data moreover suggests that chromatin and nucleosome remodellers influence homologous recombination even after DNA resection, as INO80-C is required for efficient formation of presynaptic filaments independently of its prior role in DNA end resection. These findings thus indicate that DNA end resection does not eliminate the prominent function of chromatin in the control of homologous recombination.

Speaker abstracts


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The PBAF chromatin remodelling complex and maintenance of genome stability

Peter Brownlee1, Cornelia Meisenberg1, Pedro Zuazua-Villar1, Penny Jeggo2, Jessica Downs1

1 The Institute of Cancer Research, London, UK, 2 The University of Sussex, Brighton, UK

In eukaryotes, genomic DNA is packaged into the nucleus primarily by association with histone proteins to form chromatin. This structure, while necessary for compaction and chromosome segregation, is inhibitory to most processes that require access to DNA, such as transcription, replication and repair. For this reason, cells have two powerful mechanisms for manipulating the structure of chromatin; covalent modification of histones and ATP-dependent chromatin remodelling activities. Multiple chromatin modifying activities are involved in preventing genome instability by functioning to signal and repair damaged DNA, as well as to promote faithful chromosome segregation. The PBAF chromatin remodelling complex, one of two SWI/SNF complexes in mammalian cells, plays multiple roles that contribute to genome stability. PBAF contributes to sister chromatid cohesion and centromeres, and as a consequence impacts on chromosome segregation and aneuploidy. In addition, we found PBAF helps to repress transcription in the vicinity of DNA double strand breaks. We are currently investigating the molecular mechanisms by which PBAF promotes these cellular activities.

Speaker abstracts


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Chromatin replication and epigenome maintenance

Anja Groth1

1 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, DENMARK

In dividing cells, faithful duplication of the genome must be accompanied by reproduction of the chromatin landscape on newly synthesized DNA. Our research focuses on how human cells replicate chromatin and ensure transmission of genetic and epigenetic information during cell division (reviewed in Alabert and Groth, 2012; Hammond, Stromme et al., 2017). We are interested in understanding i) how histones are handled during DNA replication () in order to propagate histone-based information, ii) how DNA replication impacts on the chromatin landscape and the mechanisms involved in restoration epigenetic marks and 3D organization, and iii) the interplay between chromatin replication and genome maintenance.

We have developed Nascent Chromatin Capture (NCC) for proteomic analysis of chromatin replication (Alabert et al., 2014) and inheritance of histone modifications during cell division (Alabert et al., 2015). We have now developed quantitative genomic approaches allowing us to study, genome wide, recycling of modified parental histones and restoration of the histone modification landscape across the cell cycle. In addition, we are taking a structure-function approach to understand mechanistically how new and old histones are handled at the replication fork. We have described how MCM2, part of the replicative helicase, acts as a histone chaperone for H3-H4 tetramers at the fork (Huang et al., 2015). More recently, we revealed a dual function of the TONSL-MMS22L homologous recombination (HR) complex as a histone chaperone and a histone reader (Saredi et al., 2016). I will discuss how this work suggest a mechanism for cells to identify sister chromatids as good substrate for error-free DNA repair and our ongoing work on the principles underlying inheritance of histone modifications in dividing cells.

Speaker abstracts


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Coupling of genes and replication origins

Ehsan Pourkarimi2, James M. Bellush2,1, Iestyn Whitehouse2

1 BCMB Graduate Program, Weill Cornell Medical College, New York, USA, 2 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, USA

Fifty percent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Working in S. cerevisiae and C. elegans, we have developed methodologies to label and purify Okazaki fragments, which we have analyzed using a combination of biochemical and deep-sequencing techniques. We show that Okazaki fragments provide a record of replication fork movement, allowing us to define a high-resolution spatial and temporal map of replication fork initiation and progression throughout a eukaryotic genome.

We find that DNA replication in C. elegans embryos is strongly correlated with “active” chromatin marks and that essentially all replication initiates at gene enhancers. Importantly, origin efficiency – the likelihood an origin is used within the population – correlates with the abundance of enhancer chromatin marks, suggesting certain histone modifications may play instructive roles in origin function. Our data is consistent with a model in which the DNA replication and transcription programs are functionally interlinked. However, the relationship is complicated as active gene transcription is not required for origin function. We present data that supports the idea that replication origins are defined by chromatin features and may be propagated by epigenetic mechanisms.

Speaker abstracts


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Proffered Paper 3

Bromodomain proteins: Readers of Genome Integrity

Kyle Miller1

1 The University of Texas at Austin, Austin, TX, USA

Chromatin-based DNA damage response (DDR) pathways are vital for maintaining genome-epigenome integrity in response to DNA damage. These pathways rely on several mechanisms to regulate the structure and function of chromatin including histone modifications, chromatin modifying enzymes and chromatin associated proteins, which can markedly influence the DDR. Determining the interplay between the DDR and chromatin is fundamental for elucidating how cells maintain both epigenetic and genome integrity. We recently identified human bromodomain (BRD) containing proteins, the primary “readers” of acetylated chromatin, as integral mediators of DNA damage signaling and repair. These studies included the discovery of the BRD protein ZMYND8 as a novel DDR factor that recruits the nucleosome-remodeling and histone deacetylation (NuRD) complex to damaged chromatin where they function to modulate transcription to promote repair of DNA double-strand breaks (DSBs). We have also identified additional chromatin factors, including histone variants and their effector proteins, which participate in DSB repair. We will report our current findings and efforts to elucidate how the DDR functions within, and coordinates the activities of, the chromatin environment to maintain genome integrity.

Speaker abstracts


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Ubiqutin dependent ATPase p97 and deubiquitinating enzyme X (DUBX) regulate DNA damage response after ionising radiation

Abhay Narayan Singh1, Judith Oehler1, Ignacio Torrecilla1, Kristijan Ramadan1

1 Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK, 2 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK

Ionising radiation (IR) induces severe DNA damage leading to cell death. DNA double strand brakes (DSBs) are the most lethal DNA lesions mainly repaired by two conserved repair pathways; non-homologous end joining (NHEJ) and homologous recombination (HR). Cells activate a robust DNA damage response (DDR) after IR to ensure DNA repair. The inherent defects in one or other DDR factor make cancer cells susceptible to IR. Identifying novel DDR factors may provide an effective strategy for improved sensitivity of cancer cells to IR. DDR relies on variety of posttranslational modifications (PTMs) and ubiquitination is a crucial PTM initiated by the E3 ubiquitin ligases RNF8 and RNF168 at damage site. Our lab has recently demonstrated that the regulation of ubiquitin signal at sites of DNA damage is driven by an ubiquitin-dependent molecular segregase/unfoldase p97. To further investigate the role of p97 in DDR, we analysed the p97 interactome after IR by SILAC based mass-spectrometry approach. We identified the deubiqutinating enzyme X (DUBX) as a major cofactor of p97 after IR. Further investigation revealed that DUBX acts as a p97-dependent cofactor to regulate turnover of ubiquitinated substrates at the sites of DNA damage. Inactivation of p97 or DUBX leads to hyper-accumulation of ubiquitinated substrates and reduced 53BP1 recruitment at sites of DSBs. Consequently, it leads to impaired NHEJ, a main DSB repair pathway after IR and hyper-sensitivity of human cancer cells to IR. Taken together, this novel p97-DUBX sub-complex could be a potential druggable target to hyper-sensitise cancer cells to IR based radiotherapy.

Speaker abstracts


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Regulation and dynamics of DNA repair in a chromatin context

Haico van Attikum1

1 Department of Human Genetics, Leiden University Medical Center, NETHERLANDS

Our cells receive tens of thousands of different DNA lesions per day. Failure to repair these lesions will lead to cell death, mutations and genome instability, which contribute to human diseases such as neurodegenerative disorders and cancer. Efficient recognition and repair of DNA damage, however, is complicated by the fact that genomic DNA is packaged in chromatin. The DNA repair machinery has to circumvent this barrier to gain access to the damaged DNA and repair the lesions. By using a cross-disciplinary approach that combines cutting-edge genomics with bioinformatics, genetics, biochemistry and high-resolution microscopy, we identified and characterized several chromatin-modifying enzymes (e.g. PARP1, CHD2 and RNF168) in DNA repair. This way we uncovered several novel mechanisms that regulate the repair of DNA damage in chromatin. At the meeting, I will present some of our recent findings and indicate the current status of our work.

Speaker abstracts


28

Proffered Paper 4

Phospho-dependent recruitment of NuA4 by MRX at DNA breaks regulates RPA dynamics during resection

Xue Cheng1, Olivier Jobin-Robitaille1, Pierre Billon1, Remi Buisson1, Hengyao Niu5, Nicolas Lacoste1, Nebiyu Abshiru2, Valerie Cote1, Pierre Thibault2, Stephen Kron3, Patrick Sung5, Christopher Brandl4, Jean-Yves Masson1, Jacques Cote1

1 Laval University, Quebec City, CANADA, 2 Universite de Montreal, Montreal, CANADA, 3 University of Chicago, Chicago, USA, 4 Western University, London, CANADA, 5 Yale University, New haven, USA

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctionnal complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability as it is a key regulator of gene expression, cell proliferation, stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double strand breaks (DSBs) in order to facilitate repair, in part through local chromatin acetylation and an interplay with Rad9/53BP1 during the DNA damage response (DDR). Yet, the precise mechanism of NuA4 recruitment to DSBs remains to be defined since its previously reported interaction with gH2AX is not required for its appearance. Here, we report a stepwise recruitment of NuA4 to DSBs, first by a DNA damage-induced and phosphorylation-dependent interaction with the Xrs2 subunit of the MRX (Mre11-Rad50-Xrs2) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break, along with the single-strand DNA binding protein RPA. Finally, we demonstrate that NuA4 can acetylate and regulate the dynamics of RPA binding to single-strand DNA, hence targeting locally both histone and non-histone proteins for lysine acetylation to coordinate repair.

Speaker abstracts


29

Proffered Paper 5

Helicases and nucleases determining replication fork recovery or demise, and implications for genome stability and human disease

Benoît Falquet2,3, Ivana Murfuni2, Gizem Ölmezer2,3, Franz Enkner2, Romain Amante1,3, Ana Correia1,3, Dominique Klein2, Mohamed Bentires-Alj1,3, Ulrich Rass2

1 Department of Biomedicine, University Hospital Basel, Basel, SWITZERLAND, 2 Friedrich Miescher Institute for Biomedical Research, Basel (BS), SWITZERLAND, 3 University of Basel, Basel (BS), SWITZERLAND

Abstract withheld from publication at the request of the author.

Speaker abstracts


30

Constructing specialized, acetylated chromatin domains to promote DSB repair

Brendan Price1

1 Dana-Farber Cancer Institute, USA

The repair of DNA double-strand breaks (DSB) is a complex process requiring tight coupling between chromatin remodeling and DNA repair. DNA damage recruits chromatin remodeling complexes to the DSB where they promote exchange of histone variants and significant rewriting of the local epigenetic signature. This includes an initial transient increase in H3K9 methylation, followed by loading of the repressive HP1-KAP1 complex, creating repressive chromatin domains which extend for hundreds of kilobases from the break. However, for DSB repair to proceed, this transient repressive chromatin at the break must be dismantled. Here, we describe the role of 2 DNA damage response proteins, HJURP and BRD2, in this process. HJURP is the chaperone for the centromere-specific histone H3 family member CENPA. CENPA is confined to centromeres in normal cells, but is overexpressed and incorporated throughout the chromatin in tumor cells. Both CENPA and its chaperone, HJURP, are recruited to DSBs, leading to CENPA exchange onto nucleosomes at the site of damage. Importantly, CENPA exchange displaces repressive HP1-KAP1 complexes from the DSB, and promotes acetylation of histone H4. Acetylated H4 then recruits the bromodomain protein BRD2, which functions to protect the newly created H4Ac from HDACs and allows H4Ac to spread along the flanking chromatin. BRD2 therefore protects a restricted, acetylated chromatin environment surrounding the DSB which is essential for DSB repair. The sequential loading and release of KAP1-HP1 complexes through CENPA exchange, coupled with BRD2-dependent increases in H4 acetylation, therefore work together to create open, repair competent chromatin at DSBs.

Speaker abstracts


31

The SUMO bomb in DNA double-strand break repair

Jo Morris1, Alexander Garvin1

1 University of Birmingham, Birmingham, UK

Ubiquitin conjugation and deconjugation are critical to the signaling events that co-ordinate several aspects of DNA double-strand break repair. More recently it has become clear that the small ubiquitin- like modifier, SUMO, also plays a central role. However compared to the ubiquitin system SUMO modification has far fewer conjugation and deconjugation components. SUMOylation appears less precise and more promiscuous than ubiquitin modification to the extent that the late Stephen Jentsch described SUMO conjugation following DNA damage in yeast as a ‘SUMO spray’, conjugating the modifier to proteins and protein groups in the vicinity of damaged chromatin.

Since SUMO conjugation can promote protein-protein interactions and act as a signal for protein degradation the cell must control the potential danger that SUMOylation poses. In this presentation how this is achieved will be explored, as will the consequences of allowing the SUMO bomb to explode uncontrollably.

Speaker abstracts


32

Proffered Paper 6

A new class of ubiquitylated-histone readers involved in the response to DNA damage

Amélie Fradet-Turcotte1

1 Université Laval, Québec, CANADA

Upon DNA double-strand breaks (DSBs), a series of phosphorylation and ubiquitylation events are initiated on the chromatin surrounding the breaks. Together with histone marks already present on the chromatin, these post-translational modifications create a code that is recognized by DNA repair proteins. We are interested in understanding how the recognition of histone modifications directs the repair of DNA. At DSBs, ubiquitylation of Histone H2A by the E3-ligase RNF168 promotes both its own recruitment and the recruitment of proteins that coordinate DNA repair, such as 53BP1, RAP80, BRCA1 and RNF169. Here, I will present our recent findings on the molecular determinants that drive the interaction of RNF168, RNF169 and 53BP1 with H2A K13- and K15-ubiquitylated nucleosomes, two products of RNF168. Our structural and biochemical studies reveal that RNF168 and RNF169 interact with H2A K13/K15-ubiquitylated nucleosomes through a bi-partite module that is composed of a ubiquitin-binding motif (MIU2) and a LR motif (LRM). Furthermore, our results suggest that the LRM participates in the interaction by docking into a negatively charged surface of the nucleosome called the acidic patch. Importantly, we show that the cooperation between the MIU2 and the LRM of RNF169 enables the recognition of a variety of ubiquitylated nucleosomes, an observation that is in sharp contrast with 53BP1, which strictly interacts with H2AK15ub nucleosomes. Globally, our studies suggest that the specificities of different ubiquitylated-chromatin binding modules contribute to the differential recruitment of DNA repair proteins to RNF168-ubiquitylated chromatin.

Speaker abstracts


33

Histone H3K36 trimethylation, genome stability and cancer

Timothy Humphrey1

1 CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK

SETD2-dependent histone H3 lysine 36 trimethylation (H3K36me3) plays a central role in both maintaining genome stability and in suppressing tumorigenesis, and is frequently depleted in particular cancer types. We find this histone mark plays an important role in promoting homologous recombination (HR) repair of DNA double-strand breaks. Further, H3K36me3 also facilitates DNA replication restart following replication stress resulting from WEE1 kinase inhibition. Accordingly, H3K36me3-deficient cancers can be specifically targeted using the WEE1 inhibitor, AZD1775, which is now in clinical trials. Yet how H3K36me3 coordinates these distinct functions to maintain genome stability is unclear. Our current understanding of these roles and their exploitation in the clinic will be presented.

Speaker abstracts


34

Proffered Paper 7

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis

Ashby Morrison1

1 Stanford University, Stanford, CA, USA

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e. adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Speaker abstracts


35

DNA damage response activation in cancer and the role of non-coding RNAs

Fabrizio d’Adda di Fagagna1,2

1 IFOM, The FIRC Institute of Molecular Oncology, Milan, ITALY, 2 Istituto di Genetica Molecolare, National Research Council, Pavia, ITALY

The DNA damage response (DDR) is a signaling pathway that arrests the proliferation of cells undergoing genotoxic events to coordinate DNA repair efforts. We previously demonstrated that DDR is physiologically activated in cancer initiation (Di Micco et al Nature 2006) and ageing (d’Adda di Fagagna et al. Nature 2003, Fumagalli et al. Nature Cell Biology 2012). Recently, we reported that a novel class of small non-coding RNA (termed DDRNA) is necessary to activate the DDR upon various sources of DNA damage (Francia et al., Nature 2012; d’Adda di Fagagna, Trends in Cell Biology 2014).

We will show that DNA double-strand breaks trigger the local transcription of the damaged locus by RNA polymerase II and that full DDR activation depends on a network of site-specific RNA:RNA interactions.

This discovery allowed us to demonstrate that sequence-specific oligonucleotides designed against non coding-transcripts generated at a damaged genomic locus inhibit DDR activation both in cultured cells and in vivo in relevant animal models.

Speaker abstracts


36

Shaping chromatin in the nucleus, the bricks and the architects

Geneviève Almouzni11 Institut Curie, Research Center ; CNRS UMR3664, Paris, FRANCE

Chromatin organization in the nucleus provides a large repertoire of information in addition to that encoded genetically. A major goal for my group involves understanding how histones, the major protein components of chromatin, the bricks, can mark functional regions of the genome through their variants or post-translational modifications, along with non-coding RNA and other chromatin regulators. Errors in the establishment and propagation of these chromatin components, possibly involving imbalance in their deposition pathways, can lead to mis-regulation of genome functions and pathological outcomes, such as cancer. The propagation of centromeric identity represents a model of choice for the study of epigenetic mechanisms. Our work has focused on histone chaperones, as architects of chromatin organisation. We will present our latest findings.

1. Maison C., Bailly D., Quivy J.P. & Almouzni G. (2016) The methyltransferase Suv39h1 links the SUMO pathway to HP1alpha marking at perticentric heterochromatin. Nature Commun., 7, 12224. doi: 10.1038/ncomms12224.

2. Adam S., Dabin J., Chevallier O., Leroy O., Baldeyron C., Corpet A., Lomonte P., Renaud O., Almouzni G. & Polo S.E. (2016) Real-time tracking of parental histones reveals their contribution to chromatin integrity following DNA damage. Mol. Cell, 64, 1-14.

3. Maison C., Quivy J.P. & Almouzni G. (2016) Suv39h1 links the SUMO pathway to constitutive heterochromatin. Mol Cell Onc., 3, 1225546, http://dx.doi.org/10.1080/23723556.2016.122554

4. Müller S. & Almouzni G. (2017) Chromatin dynamics during the cell cycle at centromeres. Nature Rev. Genet., 18, 192-208.

5. Filipescu D., Naughtin M., Podsypanina K., Lejour V., Wilson L., Gurard-Levin Z.A., Orsi G.A., Simeonova I., Toufektchan A., Attardi L.D., Toledo F. & Almouzni G. (2017) Essential role for centromeric factors following p53 loss and oncogenic transformation. Genes & Dev., 31, 463-480.

Speaker abstracts


37

INO80 chromatin remodeller links RNA quality control to transcriptional regulation

Sara Luzzi1, Camille Gautier2, Sarah Greener1, Kang Hoo Han3, B. Franklin Pugh3, Antonin Morillon2, Manolis Papamichos-Chronakis1

1 ICaMB/ Newcastle University, Newcastle upon Tyne, UK, 2 Institut Curie, PSL Research University, CNRS UMR 3244, Paris, FRANCE, 3 Pennsylvania State University, Pennsylvania , USA

Pervasive, dysregulated transcription is a source of genotoxic stress. RNA quality control mechanisms are essential for the elimination of aberrant, non-functional transcripts in order to prevent inappropriate RNA expression. Evidence indicates that mRNAs are subjected to RNA surveillance and early termination similar to non-coding RNAs, but the mechanism for removal of unproductive nascent mRNAs from chromatin remains unclear. Here we show that in budding yeast, the evolutionary conserved ATP-dependent chromatin remodelling complex INO80 globally restricts the expression of aberrant, non-productive mRNA transcripts. INO80 is enriched at the transcription start sites (TSS) across the yeast genome and at active gene promoters and enhancers in mammalian cells. INO80 promotes initiation of transcription, whilst it represses expression of ncRNAs. Although the importance of INO80 in transcription genome-wide is well established, its role remains unclear. Using recently developed high-resolution genomic techniques we discovered that loss of INO80 leads to increased promoter proximal pausing of elongating RNAPII and accumulation of nascent, aberrant mRNA transcripts on chromatin. Yeast cells lacking INO80 show marked transcriptional elongation defects, indicating that INO80 regulates transcription at a post-initiation step. Notably, enrichment of unstable transcripts on chromatin in the absence of INO80 is associated with nucleosome occupancy. We propose that chromatin remodelling by INO80 facilitates early termination of aberrant transcripts to promote productive mRNA transcription.

Speaker abstracts


38

Replication stress in chromatin: getting fragile sites in shape

Philipp Oberdoerffer1

1 National Cancer Institute/NIH, Bethesda, USA

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for gene regulation, cellular differentiation and accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at genomic regions of recurrent replication stress. Upon aberrant fork stalling within these fragile regions, DNA damage signaling and concomitant H2AX phosphorylation coordinate histone chaperone-dependent deposition of macroH2A1.2, a histone H2A variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from replication stress-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of replication stress. Altogether, our findings demonstrate that recurrent DNA damage critically contributes to the chromatin landscape to ensure both the epigenetic and functional integrity of dividing cells.

Speaker abstracts


39

Proffered Paper 8

Imprinted genes as a model system to study the impact of chromatin on targeted mutagenesis

Irene Kallimasioti-Pazi2, Gillian Taylor2, Keerthi Chathoth2, Alison Meynert2, Tracy Ballinger2, Ildem Sanli1, Sébastien Lalevée1, Robert Feil1, Andrew Wood2

1 Montpellier Institute of Molecular Genetics (IGMM), CNRS UMR-5535 and University of Montpellier, Montpellier, FRANCE, 2 MRC Human Genetics Unit, University of Edinburgh, Edinburgh, UK

Genome editing technologies allow precise modification of DNA sequences inside living cells, and are widely regarded as among the most significant biomedical research developments of the early 21st century. Genome editing nucleases introduce targeted double strand breaks, which are repaired either via non-homologous end-joining (NHEJ) generating InDels, or via homology-directed repair (HDR) pathways to produce precise sequence edits. How local variation in chromatin structure influences overall mutagenesis, and the relative contribution of NHEJ and HDR to mutagenic repair, is incompletely understood. Using CRISPR/Cas9, we have introduced double strand breaks at imprinted regions of the mouse genome, which exist naturally in divergent chromatin states on maternal and paternal chromosomes. Quantitive analysis using high throughput amplicon sequencing enabled comparisons of genome editing efficiency and mutagenic repair between allelic DNA sequences in distinct chromatin environments in the same cell nucleus. Upon prolonged exposure to Cas9, we find that active and silent imprinted alleles undergo mutations at similar frequency, whereas active alleles are up to 5-fold more frequently mutated upon short exposure or low Cas9 expression. Mutations arising from NHEJ and HDR appear with similar kinetics, and at similar frequency on silenced compared to active promoters of imprinted genes, and on transcribed versus silent gene bodies. Lastly, we find that inter-homolog recombination is rare following Cas9 cleavage in mouse embryonic stem cells. We propose that genomic imprinting and X inactivation could provide useful systems for future studies of chromatin regulation during DNA damage and repair.

Speaker abstracts


40

Proffered Paper 9

Epigenome maintenance in response to DNA damage

Sophie Polo1

1 Epigenetics & Cell Fate Centre, UMR 7216 CNRS/Université Paris Diderot, Paris, FRANCE

The response to DNA damage in the cell nucleus proceeds on a chromatin substrate, whose integrity is central to cell functions and identity. The coordinated maintenance of genome stability and of its organization into chromatin when challenged by genotoxic stress is thus critical. Yet, the underlying mechanisms are largely unknown and how much the DNA damage response impacts the chromatin landscape is poorly understood. We approach these issues by investigating alterations in histone variant patterns at sites of DNA damage in mammalian cells. By combining in vivo tracking of newly synthesized histones and localized UVC damage, we have uncovered histone deposition pathways involved in restoring chromatin structure and transcriptional activity in response to genotoxic stress (1). We have also set up an innovative system allowing simultaneous visualization of new and parental histone dynamics at sites of DNA damage in live cells, providing interesting insights into how the original information conveyed by chromatin can be preserved (2). I will present our latest findings on these topics and discuss their implications for the maintenance of chromatin integrity following DNA damage.

1. Adam, Polo*, Almouzni*. Cell, 155:94-106, 2013.

2. Adam*, Dabin*, Chevallier, Leroy, Baldeyron, Corpet, Lomonte, Renaud, Almouzni, Polo. Mol Cell, 64:65-78, 2016.

Speaker abstracts


41

Chromatin and chromosome dynamics during DNA double strand break repair

Francois Aymard1, Emmanuelle Guillou1, Marion Aguirrebengoa1, Biola Javierre2, Peter Fraser2, Gaelle Legube1

1 CBI_ LBCMCP_University of Toulouse, Toulouse, FRANCE, 2 Nuclear Dynamics Programme, The Babraham Institute, Cambridge, UK

DNA double-strand breaks (DSBs) are highly toxic lesions that are rapidly repaired by two main pathways, namely Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). Using a cell line, called DIvA (for DSB Inducible via AsiSI), where multiples breaks can be induced at annotated positions throughout the human genome, we previously reported that DSBs induced in transcriptionally active genes are channeled to HR during G2, thanks to a chromatin dependent signaling, while DSB occurring in intergenic regions rather undergo NHEJ [1]. More recently, using Capture Hi-C and high resolution microscopy, we found these DSBs induced in active genes undergo clustering in an ATM dependent manner [2] and mostly during G1 [3]. Notably, we found that clustering coincides with delayed repair in G1 (3). Collectively our data suggest that when damaged, transcriptionally active units adopt a very peculiar behavior, being repaired by HR in G2 and left unrepaired and clustered in G1.

[1] Aymard F, et al. Transcriptionally active chromatin channels DNA double strand breaks to homologous recombination. Nature structural & molecular biology. 2014 21(4):366-74

[2] Caron P, et al. Non redundant functions of ATM and DNAPK in response to DNA Double-Strand Breaks. Cell Reports. 2015 Nov 24;13(8):1598-609

[3] Aymard F, et al. Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes. Nature structural & molecular biology. 2017 24(4):353-61

This project DIvA has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 647344).

Speaker abstracts


42

Remodelers in response to DNA damage

Christian Gerhold1, Jerome Poli1, Anais Cheblal1, Andrew Seeber1, Kenji Shimada1, Susan M. Gasser1

1 Friedrich Miescher Institute for Biomedical Research, Basel, SWITZERLAND

Nucleosomes are essential for proper chromatin organization and the maintenance of genome integrity. Histones are post-translationally modified and often evicted at sites of DNA breaks, facilitating recruitment of repair factors. This action is mediated by chromatin remodelers, which evict, remodel and reassemble chromatin at sites of damage, transcription and replication. Using a range of quantitative methods we show that histone levels drop by 20-40% in response to extensive DNA damage, reflecting nucleosome eviction by the INO80 remodeler and degradation by the proteasome. Chromatin decompaction and increased fiber flexibility accompany histone degradation, but also occur in the absence of damage when histone levels are reduced by other means. In the case of DNA damage, the remodeling, histone loss and decompaction of chromatin is strictly dependent on activation of the DNA damage checkpoint kinase, ATR/Mec1. Its activity is amplified by the downstream kinase, Rad53, to provoke chromatin changes genome-wide. Changes in the physical properties of chromatin in response to DNA damage can be monitored by time-lapse imaging and a statistical analysis of single particle tracking. We analysed the chromatin locus dynamics at an inducible DSB in yeast and at other tagged loci, which all increase mobility in response to DNA damage. Polymer modeling based on the biophysical parameters of movement extracted from the imaging tracks, predicts chromatin expansion and DSB extrusion from the chromatin domain. We can differentiate between changes in extrinsic forces that drive DNA movement (arising from the cytoskeleton) and intrinsic forces that change (reflecting alterations in local chromatin structure thanks to the action of nucleosome remodelers). Finally we show that strand exchange with ectopic homology at an induced DSB, as well as gene conversion mediated by recombination are both enhanced in a manner dependent on INO80. Both correlate with enhanced movement. Thus, we propose that a generalized reduction in nucleosome occupancy is an integral part of the DNA damage response, providing mechanisms for enhanced chromatin mobility which appears to facilitate homology search.

Speaker abstracts


43

Poster abstracts

1

Chromatin-associated ARH3 is a serine mono-ADP-ribosylhydrolase

Jeannette Abplanalp2,5, Mario Leutert2,5, Emilie Frugier1, Kapila Gunasekera2, Kathrin Nowak2,5, Roxane Feurer2, Jiro Kato3, Hans A.V. Kistemaker4, Dmitri V. Filippov4, Joel Moss3, Amedeo Caflish1, Michael O. Hottiger2

1 Department of Biochemistry, University of Zurich, Zürich, SWITZERLAND, 2 Department of Molecular Mechanisms of Disease, University of Zurich, Zürich, SWITZERLAND, 3 Laboratory of Translational Research, National Heart, Lung, and Blood Institute, NIH, Bethesda, USA, 4 Leiden Institute of Chemistry Department of Bio-organic Synthesis, Leiden University, Leiden, NETHERLANDS, 5 Molecular Life Science PhD Program of the Life Science Zurich Graduate School, Zürich, SWITZERLAND

ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine were missing. Here, we report that in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometry approach showed that ARH3-deficient MEFs are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen-peroxide stress. Moreover, ARH3 was found to be associated with chromatin, and confirmed to hydrolyze ADP-ribose from histone tails and HMGB1 that were transmodified by ARTD1. We observed a global decrease of H3K27me3 in ARH3-deficient MEFs. Consequently, RNA-seq. experiments revealed major gene expression changes comparing wildtype and ARH3-deficient MEFs. The implications of these findings will be discussed.

Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress induced ADP-ribosylome as well as of the chromatin ADP-ribosylome.

Poster abstracts


44

2

CDYL1 fosters double-strand break-induced transcription silencing and promotes homology-directed repair

Enas R Abu-Zhayia1, Samah W. Awwad1, Bella Ben-Oz1, Hanan Khoury-Haddad 1, Nabieh Ayoub1

1 Technion - Israel Institute of Technology, Haifa, ISRAEL

Abnormal DNA damage response (DDR) causes accumulation of poisonous mutations that can trigger genetic instabilities and carcinogenesis. DNA damage induction is accompanied by transient transcription repression. Here, we will describe a previously unrecognized role of CDYL1 in fortifying damage-induced transcription repression and double-strand break (DSB) repair. CDYL1 is rapidly and preferentially recruited to damaged euchromatic regions. While the ECH domain of CDYL1 mediates its accumulation at DNA damage sites, the chromodomain and the H3K9me3 mark are dispensable for its recruitment. Moreover, PARP1 activity, but not ATM, regulates CDYL1 recruitment to DNA damage sites. Furthermore, CDYL1 stimulates EZH2 binding and local increase of the repressive methyl mark, H3K27me3, at damaged DNA and promotes transcription silencing at DSB sites. In addition, following DNA damage induction, CDYL1 depletion causes persistent G2/M arrest and alters H2AX and RPA2 phosphorylation. Remarkably, using the “traffic-light reporter” system, we observed that CDYL1 promotes homology-directed repair (HDR) of DSBs in vivo. Consequently, CDYL1 knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin. Altogether, we identified CDYL1 as a new component of DDR and suggest that the HDR-defective phenotype of CDYL1-deficient cells could be exploited for eradicating cancer cells harboring CDYL1 mutations.


45

Poster abstracts

3

Studying DNA damage and DNA repair in human chromatin using high-resolution genomics.

Jinchuan Hu2, Ogun Adebali2, Jason Lieb2, Aziz Sancar2, Sheera Adar1

1 Hebrew University of Jerusalem, Jerusalem, ISRAEL, 2 The University of North Carolina, Chapel Hill, USA

DNA damages block the ability of the genome to function and can lead to mutation and cancer development. Nucleotide excision repair is the sole mechanism for removing bulky and helix-distorting damages from the human genome. For carcinogenic damages induced by ultraviolet radiation or cigarette smoke, excision repair reduces cancer risk. However, in cancers, the very same repair pathway is utilized to survive DNA adducts induced by chemotherapy treatments such as cisplatin.

While the biochemistry of nucleotide excision repair is extremely well understood, how repair is orchestrated in the nucleus, where DNA is packaged into chromatin and is the template for active transcription and replication is still unclear. To study these complex interactions, we developed two genomic methods for mapping DNA damages and DNA repair at single nucleotide resolution across the human genome. Damages-seq relies on the replication-blocking properties of the damages to precisely map their location. In eXcision Repair-seq (XR-seq) we capture the excised oligonucleotides released during repair in vivo, and subject them to high-throughput sequencing. These genome-wide maps reveal relatively uniform damage formation but highly heterogenic repair. Preferential repair is observed in actively transcribed and open chromatin regions. Conversely, repair at heterochromatic and repressed regions is relatively low. Comparing repair kinetics with existing somatic mutation data from cancer cells shows late-repaired regions are associated with a higher level of cancer-linked somatic mutations. The new genomic assays we have developed will be a powerful tool in identifying new components of genome stability, and could lead to new pathways for preventive and therapeutic cancer care.


46

4

Understanding the Antagonistic Relationship between NuA4 and Rad9 at DNA Double Strand Breaks

Salar Ahmed1, Wajid Bhat1, Olivier Jobin-Robitaille1, Jacques Côté1

1 Laval University, Quebec City, CANADA

Cells are exposed to a number of genotoxic agents which give rise to lesions in DNA leading to genome instability. Failure to repair DNA double strand breaks (DSBs) is most dangerous and cells employ two major pathways to repair DSBs, Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ).

In S. cerevisiae Rad9 (mammalian 53BP1) is recruited to the DSB through its tudor domain binding H3K79me and BRCT domain that recognizes γ-H2A. NuA4 histone acetyltransferase complex (mammalian TIP60), apart from its known role in gene regulation, also has been implicated in DSB repair, favoring HR in S/ G2 phase. NuA4 mutant cells blocks in G2 in a Rad9-dependent manner.

Previous work has shown that cells in which Rad9 is mutated or the chromatin marks required for its binding are hindered show defective G1 checkpoint arrest, implicating Rad9 to be important for blocking cells in G1 phase and favoring NHEJ. Moreover, NuA4 mutant cells show a hyper-cell cycle checkpoint arrest in G1 in response to damage, implicating NuA4 in an antagonistic function with Rad9 in DSB repair.

We show that Rad9 binding to chromatin inhibits NuA4 acetyltransferase activity in vitro. Furthermore, we detect increased recruitment of NuA4 at DSBs in rad9 and yku80 mutant cells in G1, along with increased binding of RPA, Rad52 and Rad51. We speculate that in the absence of proteins that block resection in G1, NuA4 has a direct role in an alternative repair mechanism that involves DNA-end resection.

We currently investigate if other histone marks may also influence the binding of Rad9 and/or NuA4 at the DSB, and if NuA4 targets repair factors in G1. These studies will help us establish a more complete picture of the functional entanglement of NuA4 and Rad9 at the DNA breaks, in parallel to the observations we have reported in mammals.

Poster abstracts


47

5

Up-regulation of macrophage NADPH oxidase 5 expression and reactive oxygen species production by histone acetyltransferase-dependent mechanisms in atherosclerosis

Mihaela-Loredana Antonescu2, Simona-Adriana Manea2, Horia Muresian1, Adrian Manea2, Maya Simionescu2

1 Cardiovascular Surgery Department, University Hospital of Bucharest, Bucharest, ROMANIA, 2 Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Bucharest, ROMANIA

Background: Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages (MAC) express functional NADPH oxidase 5 (Nox5) that contribute to oxidative stress in atherogenesis.

Purpose: The aim of this study was to investigate the protein expression profile of representative type A (p300) and type B (HAT1) HAT isoforms, to estimate the overall histone acetylation status in human atherosclerosis, and to investigate the role of HAT-dependent mechanisms in mediating Nox5 expression and reactive oxygen species (ROS) formation in macrophages.

Methods: Non-atherosclerotic and atherosclerotic samples obtained as discarded tissues from patients undergoing carotid endarterectomy and THP-1 monocytic cells were used. THP-1 MAC were exposed to increasing concentrations of lipopolysaccharide (LPS) for up to 24 h in the absence/presence of a HAT inhibitor. Immunohistochemistry (IHC), real-time PCR, western blot, dichlorofluorescein, luciferase gene reporter, and chromatin immunoprecipitation (ChIP) assays were employed.

Results: We found that p300 and HAT1 isoforms along with Nox5 expression are significantly up-regulated in atherosclerotic plaques derived from human carotid artery compared to non-atherosclerotic samples. IHC staining revealed that p300, HAT1, Nox5, H3K27ac, and H3K9ac levels are up-regulated in CD68+ MAC-rich area within human carotid artery atherosclerotic plaques. LPS treatment induced Nox5, HAT1, H3K9ac levels, and ROS production in THP-1 Mac. Transient overexpression of p300/HAT1 enhanced the Nox5 gene promoter activity. Physical interaction of p300 and HAT1 proteins with Nox5 gene promoter at the sites of active transcription was confirmed by ChIP assays.

Conclusion: Our study provides the evidence that the histone acetylation system is altered in human atherosclerosis. Members of the HAT family control Nox5 expression and ROS production in human MAC. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.

Acknowledgements: Work supported by Romanian Academy and grants of ANCSI (PN-III-P4-ID-PCE-2016-0665 and PN-III-P2-2.1-PED-2016-1308).

Poster abstracts


48

6

A novel zinc finger containing protein, ZC3H14, is found at the DNA damage sites and it preserves genome integrity by providing efficient histones ubiquitination

Marta Baldascini1,1, Noel Francis Lowndes1,1

1 National University of Ireland NUIG, Galway, EIRE

ZC3H14 is a member of the C3H family of Zinc finger proteins. It has been previously identified as a Polyadenosine RNA-binding protein, in particular, it has been shown to regulate nuclear processing and retention of the ATP5G1 pre-mRNA (Wigington CP et al.,2016). Additionally, it has also been established that mutations in the ZC3H14 gene cause a nonsyndromic autosomal recessive form of intellectual disability due to its important role as a RNA poly(A) tail length regulator (Rha J. Et al., 2017). In this study, we identify a new role for ZC3H14 in the DNA damage response pathway (DDR). ZC3H14 was previously identified by our laboratory in a quatitative proteomic screen as an interacting partner of the central regulator of the biological responses to DNA double strand breaks (DSBs). ZC3H14 accumulates at DSBs and its depletion results in sensitivity to ionising radiation (IR), as well as persistent γH2AX and pATM(S1981) in IR-induced Foci (IRIF). These data suggest a role for this zinc finger containing protein in the DDR. Further investigation revealed a specific role for ZC3H14 in the non-homologous end joining (NHEJ) pathway of DSB repair. Consistent with such a role we observed defective recruitment of both 53BP1 and RIF1 upon depletion of ZC3H14. Furthermore, ZC3H14 is also required for the ubiquitination at sites of DNA damage that is required in turn for 53BP1 recruitment. In addition, to its C-terminal zinc finger domain, ZC3H14 also contains a centrally located Piwi-like domain (aa172-198). However, only the zinc finger domain is required for 53BP1 foci formation upon damage (IR). Taken together the data above suggests a novel role for ZC3H14 in the efficient repair of DNA double strand breaks via regulation of the ubquitiniation required for recruitment of 53BP1 to sites of DNA damage.

Poster abstracts


49

7

Screen for microRNA and drug interactions in breast cancer cell lines points to miR-126 as a modulator of CDK4/6 and PI3K inhibitors

Federica Baldassari2, Carlotta Zerbinati2, Marco Galasso2, Fabio Corrà2, Linda Minotti2, Chiara Agnoletto2, Maurizio Previati2, Carlo Maria Croce1, Stefano Volinia2

1 Ohio State University Wexner Medical Center, Columbus, Ohio, USA, 2 University of Ferrara, Ferrara, ITALY

Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments towards personalized medicine. Additionally, drug resistance is a common phenomenon. We thoroughly investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR).

Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. After this initial selection step, 4 miRNAs with the strongest impact on viability were assayed in combination with 14 BC drugs. Mann-Whitney U test with Bonferroni correction was used for statistical analysis.

Results: In a miRNA only pre-screen we observed effects on BC cell lines’ viability for 34 out of 38 candidate miRNAs, selected bioinformatically. We then identified 14 miRNA/drug combinations for which the combination IC50 was lower than that of the drug as single agent. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (paired with LEE011 and BYL719) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2+). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using HTA GeneChips and found that exogenous expression of miR-126 in BC cell lines affected the M phase of cell cycle.

Conclusion: Our results show that a combination treatment with two miRNAs, miR-181a and miR-126 enhance the activity of drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, based on a transcriptome study on miRNA treated cell lines we could pinpoint a role for miR-126 in mitotic regulation.

Poster abstracts


50

8

LINE-1 ORF1 protein is upregulated by reactive oxygen species and associated with bladder urothelial carcinoma progression

Patcharawalai Whongsiri2, Chaowat Pimratana6, Udomsak Wijitsettakul6, Depicha Jindatip1, Anapat Sanpavat3, Wolfgang Schulz5, Michèle Hoffmann5, Wolfgang Goering4, Chanchai Boonla2

1 Chulalongkorn University, Faculty of Medicine, Department of Anatomy, Bangkok, THAILAND, 2 Chulalongkorn University, Faculty of Medicine, Department of Biochemistry, Bangkok, THAILAND, 3 Chulalongkorn University, Faculty of Medicine, Department of Pathology, Bangkok, THAILAND, 4 Departments of Pathology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Düsseldorf, GERMANY, 5 Departments of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Düsseldorf, GERMANY, 6 Division of Urology, Buriram Hospital, Buriram, THAILAND

We previously demonstrated that reactive oxygen species (ROS) was a cause of Long Interspersed Nuclear Element-1 (LINE-1) hypomethylation in bladder cancer cells, and hypothesized that ROS might reactivate LINE-1 elements via epigenetic control. In this study, we investigated expression and clinical relevance of LINE-1-encoded protein (ORF1p) and oxidative stress marker 4-hydroxynonenal (4-HNE) in human bladder cancer tissues. The effect of ROS on ORF1p expression in bladder cancer cell lines was also explored. ORF1p expression and 4-HNE formation was significantly higher in cancerous tissues than adjacent noncancerous bladder tissues. ORF1p expression was positively correlated to 4-HNE expression. ORF1p expression was significantly higher in muscle-invasive tumors than in papillary/non muscle-invasive tumors and in high grade tumors compared to low grade tumors. H2O2 provoked oxidative stress and significantly upregulated ORF1p expression in the VM-CUB-1 cells compared with the untreated control, to a lesser degree in TCCSUP cells, but not in UM-UC-3 cells, corresponding to their basal level of LINE-1 hypomethylation. Co-treatment with antioxidants (tocopheryl acetate and S-adenosylmethionine) significantly inhibited ROS-induced ORF1p expression in VM-CUB-1 and TCCSUP. Treatment with H2O2 increased migration of UM-UC-3, TCCSUP and VM-CUB-1 cells; this increase was inhibited by tocopheryl acetate. In conclusion, robust histological evidence of increased ORF1p expression and 4-HNE formation in bladder cancer tissues was obtained. Elevated ORF1p expression was associated with bladder tumor progression. ORF1p expression and cell migration were experimentally increased by ROS in bladder cancer cells, suggesting that ROS could enhance LINE-1 reactivation and promote tumor progression. Attenuation of ROS generation and suppression of LINE-1 protein expression might be clinically useful to prevent bladder cancer progression.

Poster abstracts


51

9

Novel targeted small molecule inhibitors (lysine acetyltransferase inhibitors) are an exciting new selective treatment for breast cancer

Andrew McGuire3, Marie Caitlin Casey3, Alia Shalaby2, Olga Kalinina5, Emma Holian5, Catherine Curran3, Mark Webber2, Martin Scobie4, Leif Eriksson1, Michael Kerin3, Emer Bourke2, James Brown3

1 Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, SWEDEN, 2 Discipline of Pathology, National University of Ireland Galway, Galway, EIRE, 3 Discipline of Surgery, School of Medicine, National University of Ireland Galway, Galway, EIRE, 4 Division of Translational Medicine and Chemical Biology, Department of Medicine, Karolinska Institute, Stockholm, SWEDEN, 5 School of Mathematics; Statistics and Applied Mathematics, National University of Ireland, Galway, EIRE

Currently, 1 in 8 women will be diagnosed with Breast Cancer, with >500,000 deaths annually worldwide. Despite the development of targeted treatments for breast cancer linked to specific biomarkers (precision medicine), there is still a 22% mortality rate, as many breast cancers are either unaffected or develop resistance. New treatment strategies and options are urgently needed, particularly targeting resistant or late stage cancers.

We identified Tip60, an essential acetyltransferase and key mediator of the DNA damage response and repair pathway, as a druggable target. Our goal: To inhibit activity of an essential protein (with low protein levels in cancer cells) below a crucial survival threshold using small molecule inhibitors, inducing selective death in cancer cells (but not normal cells with high protein levels).

Using in silico modelling we developed novel lysine (K) acetyltransferase inhibitors (KATi) targeting Tip60. The activity of the compounds was validated using in vitro acetylation assays and on cultured breast cancer cells. The 1st generation KATi (TH1834) significantly inhibits Tip60 activity in vitro, and treating cells with TH1834 results in targeted apoptosis and increased unrepaired DNA damage (following ionizing radiation) in breast cancer, but not control, cell lines. TH1834 did not affect the activity of the highly related acetyltransferase hMOF, demonstrating specificity. Using our KATi (1st and 2nd generation) we are exploring Tip60 dependent molecular mechanisms underpinning genome stability, DNA repair and epigenetics.

Our targeted KATi are an exciting new precision medicine-based approach, selectively affecting cancer cells, and targeting hard to treat breast cancer subtypes.

Poster abstracts


52

10

Specific inhibition of chromatin modifiers impairs the recruitment of DNA repair proteins at DNA damage sites

Ignacio Campillo-Marcos1,2, Raúl García-González1,2, Pedro A. Lazo1,2

1 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Salamanca, SPAIN, 2 Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, SPAIN

Double-strand breaks (DSBs) are the most deleterious form of DNA damage and they can arise from external and internal sources. In order to preserve their genome integrity, eukaryotic cells have developed DNA damage signaling and repair machineries whose deployment depends on the nature of the lesion.

DNA damage takes place within the complex organization of chromatin, which acts as a barrier to the efficient detection and repair of DNA lesions. Because of that, all eukaryotic DSB repair pathways involve physiological chromatin alterations that facilitate the accessibility of repair machinery at damage sites and restore its local architecture afterwards. The current challenge is to understand how DNA repair occurs in the context of a highly organized chromatin environment.

During DNA damage sensing and repair, histones undergo a set of posttranslational modifications (PTMs), including acetylation and methylation. Our aim is to determine how PTMs coordinate and amplify the DNA damage response (DDR). For this purpose, different tumor cell lines were incubated with pharmacological inhibitors of chromatin modifiers, followed by induction of DNA damage by either ionizing radiation or chemotherapeutic drugs like Olaparib. Next, DNA repair foci formation was assessed, focusing on sensor and mediator proteins, such as γH2AX, Nbs1, MDC1 or 53BP1. Interestingly, 53BP1 foci formation was impaired after inhibiting histone acetylases and/or methylases, while the assembly of γH2AX ones was not affected. This supports the idea that precise PMTs are absolutely essential to 53BP1 recruitment and, thus, to DNA repair. Based on these results, we hypothesize that specific inhibitors against epigenetic marks could have potential as anti-cancer therapies, since inaccurate and inefficient repair of DSBs and changes in acetylation/methylation states would contribute to cell death in tumors.

Poster abstracts


53

11

Regulation of Ku70/80 dependent non-homologous end-joining through WWP2 mediated RPB1 ubiquitination

Pierre Caron1, Wouter W. Wiegant1, Max A.X. Tollenaere1, Angela Helfricht1, Albert Pastink1, Anton de Groot1, Martijn S. Luijsterburg1, Haico van Attikum1

1 Leiden University Medical Center, Leiden, NETHERLANDS

Protein ubiquitination in response to DNA double strands breaks (DSBs) is a key process that ensures the proper functioning of repair factors at break sites. Emerging evidence highlights important roles for RING-type E3 ubiquitin ligases in the recruitment (e.g. 53BP1, BRCA1 and XRCC4), as well as their release from DSBs (e.g. Ku70/80 and DNA-PKcs). However, the impact of the E3 ubiquitin ligase belonging to the HECT superfamily in response to DSBs is largely unknown. A genetic screen in C. elegans identified the HECT-E3 ubiquitin ligase WWP2 as an enzyme that protects cells against ionising radiation-induced DSBs. In line with this finding, we observed that WWP2 depletion in human cells increases radiosensitivity. Interestingly, we found that WWP2 accumulates at DSBs induced by UV-A laser micro-irradiation. Quantitative mass spectrometry and co-immunoprecipitation experiments revealed that WWP2 interacts with several subunits of the RNA polymerase II (RNAPII) complex, including RPB1. Further analysis showed that WWP2 depletion 1) impairs basal level of RPB1 ubiquitination and 2) reduces the transcription rate. We then want to know if WWP2 regulates transcription in response to DSBs. In response to DSBs transcription is silenced in cis and is required to promote the recruitment of Ku70/80 and the assembly of the DNA-PK complex to the broken ends. Using different methods, our first observations indicate that WWP2 participates to the transcription arrest in response to DSBs. We are verifying if WWP2 ubiquitinates RPB1 in response to DSBs. Moreover, we found that the loss of WWP2 substantially reduces the accumulation of Ku70/80, p-DNA-PKcs and XRCC4 at DSBs. Finally, we found that WWP2 depletion reduces the efficiency of repair via cNHEJ in EJ5-GFP reporter and in random plasmid integration assays. Together our data strongly suggest that WWP2 promotes Ku70/80- cNHEJ through RPB1 ubiquitination mediated transcription arrest in response to DSBs.

Poster abstracts


54

12

The Identification of Proteins and Nucleic Acids Bound to a Single Genomic Locus of Interest using enChIP

Adam Blattler1, Hodaka Fujii2, Sarantis Chlamydas1

1 Active Motif, Carlsbad,CA, USA, 2 Osaka University, Osaka, JAPAN

The advent of the CRISPR/Cas9 technology has revolutionized genome engineering resulting in a vast array of new applications based on the ability of a guide RNA (gRNA) to target a Cas9 protein in living cells, including site-directed genome editing and site-specific recruitment of modifying proteins. Here we present engineered DNA-binding molecule-mediated chromatin immunoprecipitation, or enChIP, a technique, which enables the direct study of the molecular landscape of specific genomic loci. In enChIP, a gRNA is designed to target a specific genomic sequence and when co-expressed with an epitope-tagged deactivated Cas9 (dCas9), it targets the tagged-dCas9 to a specific genomic region enabling immunoprecipitation of the locus with a tag-specific antibody. This approach provides a tool for identifying DNA, RNA and proteins that interact with a specific genomic region without prior information as to what those interacting components are, and thus has the potential to yield a comprehensive understanding of locus specific genomic regulation. Additionally, enChIP can be used to determine the specificity of a gRNA intended for use in genome engineering experiments. Here, we demonstrate enChIP’s ability to target and pull down a single genomic locus and identify inter- and intrachromosomal looping interactions associated with a CTCF binding site in human cells.

Poster abstracts


55

13

BRD3 as a specific vulnerable therapeutic target in neuroblastoma

Jolien De Wyn1, Kaat Durinck1, Anneleen Beckers1, Siebe Loontiens1, Suzanne Vanhauwaert1, Daniel Carter2, Belamy Chueng2, Glenn Marshall2,4, Katleen De Preter1, Frank Westermann3, Frank Speleman1

1 Center for Medical Genetics, Ghent University, Gent, BELGIUM, 2 Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of New South Wales, Sydney, AUSTRALIA, 3 DKFZ, Neuroblastoma Genomics, Heidelbergh, GERMANY, 4 Kids Cancer Centre, Sydney Children’s Hospital, Randwick, AUSTRALIA

Introduction: BET inhibitors have raised high expectations for cancer treatment given their anti-proliferative effect by inhibiting BRD4 and enhancer activity of highly transcribed genes such as MYC(N). However, current inhibitors also target BRD2 and BRD3 which are not functionally redundant with BRD4 and in neuroblastoma only MYCN amplified tumors respond well to these drugs.

Methods: We performed an integrated cross species bioinformatic analysis to identify candidate epigenetic regulators as targets for novel therapies in neuroblastoma.

Results: Using time-resolved expression data analysis of hyperplastic lesions and tumors from TH-MYCN transgenic mice, we first confirmed dynamic regulation of established neuroblastoma oncogenes and tumor suppressor genes. Next, we filtered within the highest upregulated genes for Cancer Gene Census (CGC) genes and identified 21 upregulated CGC genes mainly involved in chromatin remodeling and DNA repair. Finally, after further selection based on expression in CCLE and survival in neuroblastoma patients, BRD3 was identified as the top-ranked candidate: BRD3 exhibits strong upregulation during tumor formation, elevated expression is associated with very poor prognosis and BRD3 is the highest expressed in neuroblastoma in the CCLE cell line panel. We are preparing stable knockdown of BRD3 in neuroblastoma cell lines to perform RNA-sequencing. We will compare it to the downstream effects on the transcriptome as well as the impact on cell viability upon knockdown of BRD4. In addition, we dissected the BRD3 protein complex by means of label-free mass spectrometry analysis to gain further insights into the BRD3 specific functions in relation to control of gene transcription and putative interaction with transcription factors such as MYCN.

Conclusion: We identified BRD3 as a candidate novel driver gene in neuroblastoma and present our data on differential transcriptional control and protein interactions of BRD3 versus BRD4 in order to gain insight into BRD3 specific oncogenic functions.

Poster abstracts


56

14

Molecular mechanisms of transgene silencing by the Human Silencing Hub (HUSH) and MORC2

Christopher Douse1, Stuart Bloor1, Yangci Liu1, Maria Shamin1, Anna Albecka-Moreau1, Paul Lehner1, Yorgo Modis1

1 University of Cambridge, Cambridge, UK

Transcription of newly integrated transgenes is repressed by the Human Silencing Hub (HUSH) by recruitment of SETDB1 and MORC2 to the site of integration. SETDB1 deposits the repressive epigenetic mark H3K9me3 and MORC2 is an ATPase reported to have chromatin remodelling activity. Missense mutations in MORC2 have been shown to cause numerous previously-unsolved neuropathies with symptoms including severe spinal muscular atrophy.

These recent discoveries raise the following questions. How does HUSH identify target genes for silencing? How does MORC2 repress transcription? And how can MORC2 misregulation cause disease? To address these questions we have undertaken a systematic analysis of the molecular structures and biochemical properties of MORC2 and the HUSH scaffolding protein TASOR. Crystallographic and NMR data suggest that TASOR may recognize transcriptionally active loci by binding directly to nascent RNA through a domain with a poly(ADP-ribose) polymerase (PARP) fold. We have also determined structures of an N-terminal fragment of human MORC2 comprising the catalytic GHKL-type ATPase and CW-type zinc finger domains. The MORC2 N-terminus dimerizes reversibly upon ATP binding/hydrolysis to transduce HUSH silencing and contains hinged coiled coil projections not seen in related ATPases such as DNA topoisomerase II. We show that the dynamics of dimerization are perturbed in MORC2 disease variants, leading to modulation of HUSH-mediated transgene silencing activity. Our data define many essential mechanistic features of the MORC2 ATPase and provide a molecular basis for MORC2-associated neuropathies.

Poster abstracts


57

15

The BRIP1 DNA helicase is a 17q dosage sensitive cooperative driver in neuroblastoma

Suzanne Vanhauwaert2,1, Kaat Durinck2,1, Els Janssens2,1, Annelies Fieuw2,1, Bram De Wilde2,3, Genevieve Laureys3,1, Katleen De Preter2,1, Christophe Vanneste2,1, Thomas AT Look4, Frank Speleman2,1

1 Cancer Research Institute Ghent, Ghent, BELGIUM, 2 Center for Medical Genetics, Ghent University, Ghent, BELGIUM, 3 Department of Pediatric Oncology and Haematology, Ghent University, Ghent, BELGIUM, 4 Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, USA

Neuroblastoma is an aggressive pediatric tumor arising from sympathetic neuronal progenitors. Previous sequencing efforts revealed a low mutation burden while copy number alterations are highly recurrent: MYCN amplification is present in half of high risk tumors often accompanied by 1p deletions while MYCN non-amplified aggressive tumors frequently exhibit 11q deletions. Remarkably, both high risk groups show almost invariably chromosome 17q gain and we also reported that the mouse syntenic chromosome 11 region was gained during MYCN driven tumor formation. Therefore, it is likely that one or more dosage sensitive genes on 17q act as cooperative drivers during neuroblastoma development. Using an integrated bioinformatic analysis for dosage sensitive genes, we identified several candidate drivers implicated in DNA repair including BRIP1 also known as FANCJ, located on 17q23.2. BRIP1 acts as a DNA helicase in unwinding of stable G-quadruplex (G4) structures in single stranded DNA during replication ensuring timely progression through S-phase. We show that BRIP1 knock down causes increased replicative stress in MYCN overexpressing neuroblastoma cells as evidenced by increased RPA32 levels and reduced replication fork velocity. Overexpression of BRIP1 in dβh-MYCN-eGFP transgenic zebrafish caused accelerated tumor formation supporting its role as cooperative driver gene. Gene expression profiling after BRIP1 knock down confirmed enrichment for gene sets implicated in DNA replication and repair and are indicative for perturbation of G4 enriched genes. Further analysis of MYCN tumor development at multiple time points in mice revealed additional 17q genes implicated in replication fork dynamics that were upregulated during tumor formation such as BRCA1, BRCA2, EME1 and TOP2A. We therefore propose that 17q gain acts as an amplifier for increased expression of multiple genes implicated in control of replicative stress and replication fork dynamics.

Poster abstracts


58

16

The DREAM complex is a key component of MYCN driven neuroblastoma development as revealed by cross-species transcriptome analysis

Kaat Durinck2,1, Carolina Nunes2,1, Anneleen Beckers2,1, Bram De Wilde3,2, Genevieve Laureys3,2, Daniel Carter4, Belamy Chueng4, Glenn Marshall4, Christophe Van Neste2,1, Katleen De Preter2,1, Frank Speleman2,1

1 Cancer Research Institute Ghent, Ghent, BELGIUM, 2 Center for Medical Genetics, Ghent University, Ghent, BELGIUM, 3 Department of Pediatric Oncology and Haematology, Ghent University Hospital, Ghent, BELGIUM, 4 Sydney Children’s Hospital, Sydney, BELGIUM

Introduction: Neuroblastoma (NB) is a childhood malignancy with high clinical and genetic heterogeneity with poor prognosis for high risk patients, half of which exhibit MYCN amplification. MYCN driven NBs have been modeled in mouse with morphologic and genomic features closely resembling those observed in human MYCN amplified NBs. We took advantage of the power of cross-species computational analysis of genome-wide regulatory networks (interactomes) to identify master regulators controlling essential cellular processes in NB cells, in the context of MYCN driven NB tumor formation.

Results: We used the ARACNE algorithm to establish the interactome in human NB samples and applied dynamic differential expression analysis to determine patterns of transcriptional changes in the course of murine MYCN-driven tumor development using an established MYCN neuroblastoma mouse model. In a further step, we used the MARINA algorithm and identified the DREAM complex components FOXM1, MYB and EZH2 as important regulators of NB development. Based on these findings, we will test FOXM1 as a novel vulnerable target in NB by pharmacological inhibition of MELK, an FOXM1 upstream regulatory kinase, using the small molecule inhibitor (MELK-T1) and test possible synergism with MYB and EZH2 inhibition using in vitro cellular NB models and a dbh-MYCN driven zebrafish NB model.

Conclusion: We propose a model whereby MYCN cooperates with a FOXM1/DREAM complex regulatory network that can serve as a novel vulnerable node for targeted therapy development for NB patients and provides a prelude for potential synergistic drug combinations.

Poster abstracts


59

17

Re-expression of ER and HER2 by epigenetic modifiers sensitize breast cancer cells to hormonal therapy

Wafaa Ramadan3, Ekram Saleh2, Cijo Vazhappilly3, Varsha Menon3, Raafat El-Awady1,3

1 College of Pharmacy, University of Sharjah, Sharjah, UNITED ARAB EMIRATES, 2 National Cancer Institute, Cairo University, Cairo, EGYPT, 3 Sharjah Institute for Medical Research, University of Sharjah, Sharjah, UNITED ARAB EMIRATES

Triple negative breast cancer (TNBC) subtype is the most heterogeneous type, more aggressive with poorer prognosis compared to other breast cancer subtypes. It lacks estrogen and progesterone receptors expression and lacks the amplification of Human Epidermal Growth Factor Receptor 2 (HER2). So far there are no potential targeted therapy for TNBC patients. The aim of the present study is to investigate the effect of epigenetic modifiers (SAHA and 5-aza-dc) on the expression of estrogen receptor α (ERα), HER2 and on the response of different breast cancer cell lines to the estrogen receptor modulator Tamoxifen. Our results show differential basal expression of epigenetic markers including DNA methyltransferase 1 (DNMT1) and histone deacetylases (HDACs) in four breast cancer cell lines with different hormonal status (MCF7, SkBr3, BT-549 and MDA-MB-231). Treatment with epigenetic modifiers changed the expression level of ERα and HER2 in these cells. The sensitivity of these four cell lines to Tamoxifen was enhanced upon combination with epigenetic modifiers, especially the resistance of TNBC cells (BT-549 and MDA-MB-231) to Tamoxifen was overcome using this combination treatment. Moreover, SAHA and 5-aza-dc induce apoptosis through altering the expression of p53, c-MYC, Bid and Bcl-xl and changing the expression and cleavage levels of caspases 3, 9 and 8.The results of the present study indicate that modification of epigenetic status of breast cancer cells specially the triple negative ones enhances their response to hormonal therapy through upregulation of ERα and induction of apoptosis, which opens the avenue for using combination of epigenetic modifiers with hormonal therapy to overcome resistance of TNBC cells.

Poster abstracts


60

19

Identifying the substrate(s) of a conserved ubiquitin binding domain in CSB that facilitates transcription-coupled repair

Liam Gaul1, Jesper Svejstrup1

1 The Francis Crick Institute, London, UK

Cockayne syndrome protein B (CSB) is vital for transcription-coupled nucleotide excision repair (TC-NER). While CSB has been implicated in many distinct steps, from repair factor recruitment to transcription restart, its mechanism of action remains unclear. The Svejstrup lab has identified a ubiquitin binding domain (UBD) in CSB that renders TC-NER defective, while still supporting recruitment of repair factors. Stalled RNA Polymerase II (RNAPII) at sites of damage is an obstruction that must be removed for repair of lesions via an unknown mechanism. I am pursuing the hypothesis that CSB’s UBD binds ubiquitin at damage-induced ubiquitination sites elsewhere in the protein, altering its structure and activity. To study this, I am creating mutants in which known ubiquitination sites are mutated and analysing their phenotype through expression in CS patient-derived cells.

Poster abstracts


61

20

Understanding the Response to DNA Damaging Agents on Wee1 kinase Inhibition in High Grade Serous Ovarian Cancer and Investigating Mechanisms of Resistance.

Miriam Kathleen Gomez3,1, David W. Melton2, Charlie Gourley3

1 Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology., Islamabad, PAKISTAN, 2 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK, 3 The Nicola Murray Centre for Ovarian Cancer Research, Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

High grade serous ovarian cancer (HGSOC) makes up about 80% of total ovarian cancer cases and has a high mortality rate. As a consequence of p53 mutation the G1/S checkpoint is lost and HGSOCs have an increased reliance upon the G2/M checkpoint for DNA damage detection and repair. This reliance has led to interest in inhibition of the G2/M checkpoint as a therapeutic treatment for HGSOC. Wee1 kinase plays an important role at the G2/M checkpoint by phosphorylating Cdk1 and inactivating the Cdk1-cyclinB complex causing a G2 arrest. The Wee1 kinase inhibitor, AZD1775, has displayed favourable activity in a variety of pre-clinical tumour models in combination with DNA damaging agents. We assembled a panel of twelve validated HGSOC cell lines representing the different molecular subtypes of HGSOC. We then measured sensitivity changes to the DNA damaging agent cisplatin, or PARP inhibitor olaparib, in combination with AZD1775 in the HGSOC cell line panel by growth and viability assays. A considerable increase in sensitivity to the two agents was observed on addition of AZD1775 in some of the HGSOC cell lines. Since understanding acquired resistance mechanisms will be critical for developing effective Wee1 inhibitor combination therapies, we isolated clones resistant to AZD1775 alone and in combination with cisplatin. Resistant clones showed increased IC50 values for AZD1775 and cisplatin and, when exposed to AZD1775, they showed strikingly normal cell cycle profiles compared to the highly dysregulated profile of the AZD1775-exposed parental sensitive cell line. To identify the mechanisms of resistance to AZD1775 in HGSOC, we are investigating expression levels and mutational status of Wee1 kinase itself and the other players involved in the DNA damage-induced G2 arrest response.

Poster abstracts


62

21

Overexpression of Aiolos Promotes Epithelial-mesenchymal Transition through Control of Twist and matrix metalloprotease 1 in lung cancer

Jung-Jyh Hung1

1 Taipei Veterans General Hospital, Taipei, TAIWAN ROC


Poster abstracts


63

22

Impact of the Epigenetic Factor BORIS on DNA Damage Sensitivity in Cancer

Sanne Janssen4,3, Léon van Kempen4,3, Mounib Elchebly4, Alan Spatz4,2

1 Department of Oncology, McGill University, Montreal, QC, CANADA, 2 Department of Pathology and Oncology, McGill University, Montreal, QC, CANADA, 3 Department of Pathology, McGill University, Montreal, QC, CANADA, 4 Lady Davis Institute for Medical Research, Montreal, QC, CANADA

Background: Environmental insults as well as errors during DNA replication cause damage to our DNA that can lead to mutations and diseases, like cancer. Cells have evolved mechanisms to detect and repair DNA damage. However, if DNA repair fails or the amount of damage is overwhelming, the cell undergoes apoptosis or can become cancerous. DNA is more sensitive to damage when in an open, decondensed conformation. BORIS, also known as CTCF-Like, is associated with epigenetic changes that contribute to DNA decondensation and is abnormally expressed in various cancers. We hypothesize that BORIS sensitizes cells to DNA damage by promoting an open DNA conformation.

Objective: To obtain a better understanding of BORIS’s role in DNA organization and DNA damage by studying these processes in cancer cell lines with low or high BORIS expression.

Methods: BORIS protein expression was established in low BORIS expressing cell lines using a doxycycline inducible system. Flow cytometry, western-blot, crystal violet and MTT assays were performed on BORIS expressing and control cells to assess proliferation, cell cycle, DNA damage and apoptosis. DNA damage was induced by exposure to Olaparib (a PARP inhibitor) followed by proliferation and apoptosis assays. DNA conformation is being determined by ATAC-sequencing.

Results: Induction of BORIS expression resulted in a significant reduction in cell growth, increased cell death and a delay in S-phase. DNA damage marked by yH2AX was increased and we observed ATM-mediated activation of the DNA damage pathway in BORIS expressing cells. Exposing these cells to Olaparib displayed an increased level of apoptosis compared to control.

Significance: Understanding the role of BORIS in directing DNA conformation and the consequential effect on DNA damage will demonstrate if BORIS expressing cells are more prone to DNA damage, which could provide a basis for the use of DNA damaging agents in BORIS expressing cancer cells.

Poster abstracts


64

23

Epigenetic mechanisms of pericentromeric satellite-RNA expression and its association to malignant transformation

Michelle Hussong7,2, Julian Kanne7, Christina Grimm7, Christian Kähler2,5, Martin Kerick7,4, Alexandra Franz4,3, Bernd Timmermann6, Jörg Isensee1, Tim Hucho1, Stefan Börno2, Franziska Welzel5, Michal R. Schweiger7,2

1 Department of Anesthesiology and Intensive Care Medicine, Cologne, GERMANY, 2 Department of Biology, Chemistry and Pharmacy, Free University Berlin, Berlin, GERMANY, 3 Institute of Molecular Life Sciences, University of Zurich, Zurich, SWITZERLAND, 4 Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Berlin, GERMANY, 5 Max Planck Institute for Molecular Genetics, Otto-Warburg Laboratory ‘Neurodegenerative disorders’, Berlin, GERMANY, 6 Max Planck Institute for Molecular Genetics, Sequencing Core Facility, Berlin, GERMANY, 7 University Hospital of Cologne, Functional Epigenomics, Cologne, GERMANY

The highly organized human genome can be divided in the open and transcriptionally active euchromatin and the condensed and mainly transcriptionally silent heterochromatin. Constitutive heterochromatin is located on pericentromeric regions and characterized by specific DNA hypo-methylation on repetitive satellite DNA regions as well as by enrichment of H3K9me3 as well as H4K20me2/3. During malignant transformation, ageing or stress induction the pericentromeric heterochromatin decondensates and induces the expression of long non-coding satellite RNA (SATIII). The function of these pericentromeric transcripts is largely unknown. Besides a putative role as modulators of stress-induced splicing process, SATIII RNA are involved in heterochromatin formation and maintenance as well as genomic stability, making them an interesting target in cancer research. Another very promising target for cancer therapy, with widespread success in diverse tumor entities, is the bromodomain protein BRD4. BRD4 is an epigenetic reader and has gained extensive attention mainly due to its involvement in tumor growth. We discovered that BRD4 is a central regulator of the heat stress response, which shares high similarities with proteotoxic stress found in many cancer cells. Using RNA-sequencing analyses of BRD4-deficient and heat treated cells we identified BRD4 as an epigenetic factor in the heat shock-mediated splicing regulation and in the transcriptional activation of SATIII repeats. These non-coding RNAs accumulate together with the heat shock factor 1 (HSF1) and several epigenetic regulators, such as BRD4, at their genomic locus forming so called nuclear stress bodies (nSB). We have now extended our analyses and gained new insights in the epigenetic mechanism of de-regulated pericentromeric satellite DNA and in the associated splicing process in cancer and therapy resistance.

Poster abstracts


65

24

Overexpression of IL-15 and MCP-1 initiate genomic instability through the activation of the signal transducer and activator of transcription 3 (STAT3) and proto-oncogene protein MYCL1.

Daehong Kim2,1, Giljun Park2,1, Hanna Rajala2, Satu Mustjoki2,1

1 Department of Clinical Chemistry and Hematology, University of Helsinki, Helsinki, FINLAND, 2 Hematology Reserach Unit Helsinki, University of Helsinki and Department of Hematology, Comprehensive Cancer Center, Helsinki University Hospital, Helsinki, FINLAND

Proinflammatory cytokines and chemokines have been considered as a main driver for chronic inflammation, which has long been linked to both autoimmune diseases and cancer. One of the key inflammatory signalling pathway (JAK-STAT) has been found to be mutated in T-LGL leukemia (lymphoproliferation associated with autoimmune disorders and somatic STAT3 and STAT5B mutations), but detailed molecular mechanism has not been cleared yet. Here we hypothesized that genomic instability caused by constitutive overexpression of proinflammatory cytokine, IL-6, IL-15, and/or chemokine, MCP-1, could be one of the risk factors to induce somatic mutations associated with aberrant autoimmunity as well as hematological malignancy. We analyzed mRNA expression levels of IL-6, IL-15, MCP-1, and their related genes in primary CD8+T cells from T-LGL patients with quantitative real-time PCR. We found that LGL leukemia patients had increased expression of IL-15, MCP-1, IL-2RG, AURKA, AURKB, DNMT1, DNMT3B, HDAC2, HDAC8, BRCA1, and MYCL1 compared with healthy control. Immunoblot analysis showed that patients with STAT3 or STAT5B mutations had increased phosphorylation of STAT3 associated with overexpression of MYCL1 and DNMT1 in CD8+ T cells. Furthermore, exogenously and stably induced STAT3 mutations in EBV-infected NK-like cell line, KAI3 cells, induced DNMT1 and p-STAT3 overexpression. Immunoprecipitation analysis confirmed STAT3 to be in complex with DNMT1 and HDAC1 in T lymphoblast cell line SUP-T1 thus providing the mechanism for DNMT1 and HDAC1 to enter into the nucleus and bind to DNA. Additionally, exogenous IL-15 and MCP-1 treatment induced DNMT1 and EZH2 expression in healthy CD8+ T cells together with STAT3 phosphorylation. Taken together, these findings suggest that proinflammatory cytokine IL-15 or chemokine MCP-1 increase the expression of DNMT1, EZH2, and MYCL1 through STAT3-mediated signalling. The aberrant expression of DNMT1, EZH2, and MYCL1 indicate a potential genomic instability, which may result in the formation of somatic mutations.

Poster abstracts


66

25

Quantification of temporal changes in histone post-translational modifications associated with gH2AX nucleosomes using targeted mass spectrometry

Zuzanna Kozik1, Steve Sweet1

1 Genome Damage and Stability Centre, University of Sussex, Brighton, UK

DNA double strand breaks (DSBs) pose a major threat to the maintenance of genetic integrity. Cells have evolved response pathways to detect, signal and repair those lesions. Alterations in the factors involved in these pathways may lead to disease development, such as cancer.

Several epigenetic modifiers have previously been shown to be recruited to the sites of DSBs, but their role in the repair process still remains controversial. It has been proposed that the cellular response to DSBs leads to changes in methylation, acetylation and ubiquitination of histones at the site of damage. Some of these modifications are known epigenetic markers involved in maintaining cellular identity. It has been proposed that DSB-induced alterations to the epigenetic code introduce a potential window of opportunity for pathological changes to occur.

Our aim is to quantify the dynamic changes in the histone post-translational modifications (HPTMs) on the nucleosomes, which are directly associated with S139 phosphorylated histone H2AX (gH2AX), a cellular response to DSBs. To do that, we have designed and optimised chromatin immunoprecipitation (ChIP) of the mono-nucleosomes containing histone gH2AX, followed by targeted mass spectrometry (MS). The strength of this system is that it allows us to quantify multiple modifications on particular histone peptides in a single MS run. Validating our ChIP-MS approach, we were able to confirm that at least 50% of the histone H2A in our ChIP samples is H2AX and more than 90% of this H2AX is phosphorylated on S139. Additionally, we observe a robust induction of the ubiquitination of K15 on histone H2A variants (a known DSB marker) following damage, further confirming we are recovering histones from the DSB region. Contrary to previous reports, we do not see significant changes in methylation of K9 on histone H3 or acetylation of K16 on histone H4.

Poster abstracts


67

26

Yes-associated protein expression is correlated to the differentiation of prostate adenocarcinoma

Jae-Hyuk Lee1, Myung-Giun Noh1, Chan Choi1, Dong-Deuk Kown2

1 Department of Pathology/Chonnam National University Hwasun Hospital, Hwasun/Jeollanam-do, SOUTH KOREA, 2 Department of Urology/Chonnam National University Hwasun Hospital, Hwasun/Jeollanam-do, SOUTH KOREA

Background: Yes-associated protein (YAP) in the Hippo signaling pathway is a growth control pathway that regulates cell proliferation and stem cell functions. Abnormal regulation of YAP was reported in human cancers including liver, lung, breast, skin, colon and ovarian cancer. However, the function of YAP is not known in prostate adenocarcinoma. The purpose of this study was to investigate the role of YAP in tumorigenesis, differentiation of the carcinoma, and prognosis of prostate adenocarcinoma. Methods: The nuclear and cytoplasmic expression of YAP was examined in 188 cases of prostate adenocarcinoma using immunohistochemistry. YAP expression levels were evaluated in the nucleus and cytoplasm of the prostate adenocarcinoma cells and the adjacent normal prostate tissue. The presence of immunopositive tumor cells was evaluated and interpreted in comparison with the patients’ clinicopathologic data. Results: YAP expression levels were not significantly different between normal epithelial cells and prostate adenocarcinoma. However, YAP expression level was significantly higher in carcinomas with a high Gleason grade (8-10) than in carcinomas with a low Gleason grade (6-7)(p<.01). There was no statistical correlation between YAP expression and stage, age, PSA level and tumor volume. Biochemical recurrence (BCR) free survival was significantly lower in patients with high YAP expressing cancers (p=.02). However high YAP expression was not an independent prognostic factor for BCR in the Cox proportional hazards model. Conclusions: The results suggested that YAP is not associated with prostate adenocarcinoma development, but it may be associated with the differentiation stages of the adenocarcinoma. YAP was not associated with BCR.

Poster abstracts


68

27

Inherited DNA lesions determine G1 duration in the next cell cycle

Aleksandra Lezaja1, Matthias Altmeyer1

1 Department of Molecular Mechanisms of Disease, University of Zurich, CH, SWITZERLAND

Replication stress is a major source of DNA damage and an import driver of cancer development. Replication intermediates that occur upon mild forms of replication stress frequently escape cell cycle checkpoints and can be transmitted through mitosis into the next cell cycle. The consequences of such inherited DNA lesions for cell fate and survival are poorly understood. By using time-lapse microscopy and quantitative image-based cytometry to simultaneously monitor inherited DNA lesions marked by the genome caretaker protein 53BP1 and cell cycle progression we show that inheritance of 53BP1-marked lesions from the previous S-phase is associated with a prolonged G1 duration in the next cell cycle. These results suggest that the cellular heterogeneity observed at the level of G1/S transition is not stochastic but can be explained by the history of each individual cell and by how faithful and complete DNA replication occurred during the previous S-phase. The time, which a cell spends in G1, is thus determined at least in part by the amount of replication remnants that have been transmitted through mitosis. We further show that loss of the tumor suppressor protein p53 overrides replication stress-induced G1 prolongation and allows S-phase entry with excessive amounts of inherited DNA lesions. Thus, replication stress and p53 loss may synergize during cancer development by promoting cell cycle re-entry with unrepaired mutagenic DNA lesions originating from the previous cell cycle.

Poster abstracts


69

28

The histone chaperone HIRA is crucial for the early establishment of hepatitis B virus minichromosome

Maëlle Locatelli2,4, Judith Fresquet2, Sarah Maadadi2, Barbara Testoni2,4

1 Hospices Civils de Lyon (HCL), Lyon, 69002, FRANCE, 2 INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69003, FRANCE, 3 Laboratoire d’excellence (LabEx), DEVweCAN, Lyon, 69008, FRANCE, 4 University of Lyon, Université Claude-Bernard (UCBL), Lyon, 69008, FRANCE

Hepatitis B virus (HBV) covalently-closed-circular (ccc)DNA, the viral minichromosome , resides in the nucleus of infected hepatocytes by virtue of its chromatin structure. Indeed, upon entry into hepatocytes, the partially double stranded viral relaxed circular DNA (rcDNA) is released into the nucleus, where it is repaired and wrapped by histones to form an episomal chromatinized structure.

We investigated the role of host factors belonging to DNA repair and nucleosome assembly pathways in cccDNA formation at early time points (i.e. between 30 minutes and 72 hours) post-infection in both HepG2-NTCP cell line and primary human hepatocytes (PHH). We particularly focused on the histone chaperone Hira, which is known to deposit histone variant 3.3 (H3.3) onto cellular DNA in a replication-independent manner and in association to nucleosome reshuffling during transcription and DNA repair.

Knock-out of Hira by RNA interference before virus inoculation led to a strong decrease in cccDNA accumulation (which was independent from HBx protein expression (using an HBx-defective virus). rcDNA levels remained stable, indicating either a possible incomplete or delayed rcDNA to cccDNA transition. Chromatin Immunoprecipitation analysis showed that Hira was bound to cccDNA at 2h post-infection and that its recruitment was concomitant with the deposition of histone H3.3 and the binding of HBV capsid protein (HBc). H3.3 increase at 24h p.i correlated with the initiation of HBV RNAs transcription. By co-immunoprecipitation and Proximity Ligation assay experiments, we showed that Hira was able to interact with HBc in infected hepatocytes and in an HepaRG cell line expressing HBc in an inducible manner.

Altogether, our results suggest that chromatinization of incoming viral DNA is a very early event, requiring the histone chaperone Hira. The recruitment of Hira could be mediated by its interaction with HBc, suggesting that this interaction could represent a new therapeutic target to be investigated.

Poster abstracts


70

30

The ribonucleotide reductase subunit M2 (RRM2) is a druggable copy number driven dependency gene in neuroblastoma

Carolina Nunes2,1, Siebe Loontiens2,1, Pauline Depuydt2,1, Christophe Van Neste2,1, Kaat Durinck2,1, Frank Speleman2,1

1 Cancer Research Institute Ghent, Ghent, BELGIUM, 2 Center for Medical Genetics, Ghent University, Ghent, BELGIUM

Neuroblastoma (NB) is a pediatric cancer arising from the sympathetic nervous system and predominantly DNA copy number driven. Highly aggressive MYCN amplified NB also presents with large 1p deletions, distal 2p gains in addition to MYCN amplification (2p24.2) and 17q gain. To this end, we set out to identify cancer dependencies for genes affected by recurrent DNA copy number gains.

We identified the ‘ribonucleotide reductase subunit M2’ (RRM2) amongst the highest ranked candidate dosage sensitive dependency genes in NB. RRM2, encoded at 2p25.1 distal to MYCN, is an essential enzyme for DNA replication and repair. Correlation analysis for RRM2 using transcriptome data of NB patient cohorts revealed enrichment for a FOXM1 signature. These analyses also provided, in accordance with the known interaction between CHD5 and WEE1, evidence for a CHD5-WEE1-RRM2 regulatory axis in NB. Notably, recent work has indicated that BRCA1, encoded on 17q and highly expressed in NB cells, positively regulates RRM2 levels. Taken together, these findings suggest that recurrent co-occurrence of 1p deletion and 2p and 17q gains in MYCN amplified NB, collectively converge towards induction of increased RRM2 levels.

RRM2 depletion in NB cells induces reduced colony formation capacity and increased apoptosis. At the molecular level, stable downregulation of RRM2 induces a profound downregulation of a FOXM1 signature and upregulation of a TP53 signature. Next, we evaluated sensitivity of NB cells to the direct RRM2 inhibitors Hydroxyurea (HU) and Triapine as well as to MK-1775, an inhibitor for the RRM2 upstream regulator WEE1. Using a dbh-MYCN zebrafish model and mouse xenografts, we are currently further testing the in vivo effects of direct RRM2 inhibition and WEE1 inhibition.

In conclusion, we identified RRM2 as a novel druggable copy number driven dependency gene further supported by recurrent 1p, 2p and 17q copy number alterations in NB.

Poster abstracts


71

31

Gene expression profile regulated by cell-type specific enhancers in breast cancer

Sumin Oh1, Chaeun Oh1, Chaerin Ryu1, Sunyoung Jang1, Kyung Hyun Yoo1

1 Sookmyung women’s university, Seoul, SOUTH KOREA

Breast cancer is most popular disease in women worldwide. As disease causes are various in breast cancer occurring and result in different phenotypes, breast cancer is divided as particular subtypes depending on hormone receptor regulation, ER, PR, and HER2. Previous studies have been focused on discovering pathogenesis genetic mutations. Nevertheless, it is still not revealed enough to understand pathogenesis mechanism, genetically. Recently, studies have discovered also epigenetic modification can influence to alter gene regulation related with disease-causing.

The aim of this study is to detect the genetic responsible features in each subtypes of breast cancer. Global analysis of RNA-seq and ChIP-seq from GEO database was performed in representative three subtypes, MCF-7 (ER positive), SK-BR-3 (HER2 positive) and MDA-MB-231 (Triple negative breast cancer (TNBC)) cell lines. Approximately 900 genes were significantly up-regulated in MCF-7 cell line compare to other cell lines. It has been identified the expression of GATA3, SLC39A6, and GREB1 was remarkably increased in MCF-7 cells using qRT-PCR. In addition, genomic features of these genes were investigated with histone modification marks, H3K27ac (active enhancer marker) and H3K4me3 (promoter marker) as well as cell-type specific transcription factor bindings. Enrichment of transcription factor with H3K27ac was confirmed at enhancer regions corresponding with gene expression levels. These results demonstrated that cell-type specific enhancers might be involved in cell-type specific transcription regulation suggesting that could be key elements for therapeutic effect in breast cancer.

Poster abstracts


72

32

Involvement of PAF1 complex in the DNA damage response.

Garin Park1,1, Nari Kim1,1, Joo-yeon Yoo1,1

1 POSTECH, Pohang, Gyeoungbuk, SOUTH KOREA

PAF1 complex is a transcriptional complex composed of PAF1, CTR9, CDC73, LEO1, RTF1 and SKI8 in human. It plays a key role in the regulation of every step of transcription, from initiation to termination. PAF1 complex associates with RNA polymerase Ⅱ during transcription, and regulates modification patterns of surrounding histones or chromatin structure via recruitment of histone modifying enzymes or chromatin remodeling factors to the actively transcribed site. In yeast, PAF1 complex has been also proposed to involve in other DNA associated processes, like DNA recombination, replication, and repair. However, detailed molecular mechanism of this regulation has not been fully understood. We recently observed significant DNA breakage in the PAF1 complex depleted cells. DNA repair of artificially introduced DNA break by endonuclease SecI was less efficiently repaired in these cells, indicating the connection between the DNA damage repair and active transcription. The molecular mechanism of this regulation will be discussed.

Poster abstracts


73

33

Non-coding RNAs in the modulation of class switch recombination

Ali Rahjouei1, Miha Milek1, Michela Di Virgilio1, Markus Landthaler1

1 Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, GERMANY

B lymphocytes are a main component of the adaptive immune system. These cells produce antibodies, which can be either inserted into B cell plasma membrane (B cell receptor, BCR) or can be secreted (Immunoglobulin, Igs). Different classes of Igs (IgG, IgE, IgA) exert different effector functions and are defined by the constant-region of the Ig heavy chain (Igh). During the course of an immune response, B cells can alter the Igh constant region, thus expressing an antibody of same antigen specificity but different isotype. This process is known as class switch recombination (CSR) and it happens in B cells in response to different stimuli in an irreversible way. Stimulated B cells express a unique deaminase (activation-induced cytosine deaminase or AID) that targets highly repetitive regions at the Igh locus (switch region, S) in collaboration with processed long non-coding RNAs (known as germ line transcript or GLT). AID activity at S regions leads to the formation of DSBs and initiates a deletional DNA recombination reaction that relies on components of the general DNA damage response. The role of GLT in the initial steps of CSR represents one of the most studied functions of noncoding RNAs during antibody gene diversification, however, a comprehensive picture of the roles played by ncRNAs during pre and post-AID targeting events is lacking.

In this study, we propose to characterize the RNA-regulatory networks that modulate CSR. To do so, we generated a model system that would allow us to temporally control the different steps of CSR in an independent manner. This system will provide a powerful tool for the characterization of the RNA-protein interactions supporting CSR.

Poster abstracts


74

34

Role of the CHD7 chromatin remodeler in DNA double-strand break repair

Magdalena B. Rother1, Wouter W. Wiegant1, Martijn S. Luijsterburg1, Haico van Attikum1

1 Leiden University Medical Center, Leiden, NETHERLANDS

DNA is constantly exposed to damaging agents. Of all the DNA lesions that are induced, DNA double-strand breaks (DSBs) are among the most toxic since they can lead to mutations and chromosomal rearrangements, which underlie cancer development. DSBs are repaired via two main pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). Moreover, DSBs are repaired in the context of chromatin. To provide repair enzymes access to these breaks, ATP-dependent chromatin remodeling complexes remodel chromatin during the repair process. Several members of the CHD family of chromatin remodelers, including CHD1, CHD2, CHD3 and CHD4 play important roles in DSB repair. Here we identify CHD7 as a novel CHD chromatin remodeler that is rapidly and transiently recruited to DNA damage induced by laser micro-irradiation in a manner dependent on poly(ADP-ribose) polymerase (PARP) activity. Moreover, co-immunoprecipitation experiments showed that CHD7 interacts with PARP1. CHD7 depletion by siRNA and CRISPR/Cas9-based genome editing rendered cells sensitive to ionizing radiation and impaired NHEJ measured by EJ5-GFP reporter and random plasmid integration assays. In line with these results, we observed a decrease in the accumulation of the core NHEJ factor XRCC4 at laser-induced DNA damage in CHD7 depleted cells. Importantly, expression of GFP-tagged CHD7 in the CHD7 depleted cells could rescue XRCC4 accumulation. Finally, CHD7 depleted cells were not impaired in HR. Taken together, our data reveal that CHD7 promotes DSB repair via NHEJ by facilitating the assembly of the XLF-XRCC4-DNA ligase IV complex at the DSBs. Further experiments are being performed to unravel the exact mechanism of CHD7 mode-of-action during NHEJ. Additionally, CHD7 mutations cause CHARGE syndrome characterized by congenital anomalies such as malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. We will study whether the CHARGE mutations in CHD7 lead to NHEJ impairment and subsequently to the disease.

Poster abstracts


75

35

Evolutionary Analysis reveals DNA alkylation damage is a byproduct of cytosine DNA methyltransferase activity

Cristina Requena2,1, Silvana Rosic2,1, Rachel Amouroux2,1, Max Emperle3, Ana Gomes2,1, Albert Jeltsch3, Sarah Linnett2,1, Murray Selkirk1, Petra Hajkova2,1, Peter Sarkies2,1

1 Imperial College London, London, UK, 2 MRC London Institute of Medical Sciences, London, UK, 3 University of Stuttgart, London, UK

Methylation at the 5 position of cytosine in DNA (5meC), is the archetypal epigenetic mark in eukaryotes. Once introduced by de novo methyltransferases (DNMT3a/b in mammals), 5meC can be maintained through DNA replication due to the ability of a “maintenance” methyltransferase (DNMT1 in mammals). Despite their ancient origin, DNA methylation pathways differ widely across metazoans, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. Here we use comparative epigenomics across the nematode phylum to gain insight into the evolution of DNA methylation. Although the model nematode C. elegans has lost DNA methylation, more basal nematodes retain the methylation pathway, where it is targeted to repeat loci. Unexpectedly, we find that DNA methylation coevolves with the DNA alkylation repair enzyme ALKB2 across eukaryotes. We show that DNA methyltransferases cause alkylation damage in vitro and in vivo by introducing 3meC into DNA. Alkylation damage is thus a cost associated with DNA methyltransferase activity, which may drive loss of DNA methylation in many species.

Poster abstracts


76

36

Role of the Tousled like kinases in maintaining genome and epigenome stability

Sandra Segura-Bayona2, Camille Stephan-Otto Attolini2, Sung-Bau Lee1,3, Philip A Knobel2, Marina Villamor-Payà2, Anja Groth1, Travis H Stracker2

1 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, DENMARK, 2 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, SPAIN, 3 Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, College of Pharmacy, Taipei Medical University, Taipei, TAIWAN ROC


Poster abstracts


77

37

Identification of microRNA signature in different Paediatric Brain Tumours

Marwa Tantawy1, Mariam Elzayat1, Dina Yehia1, Hala Taha1,2

1 Children’s Cancer Hospital Egypt, Cairo, EGYPT, 2 National Cancer Institute, Cairo, EGYPT

Understanding paediatric brain tumour biology is essential to develop disease stratification, less toxic therapeutic agents, and to find novel markers for early diagnosis. MicroRNAs play a significant role in brain tumour biology and may be up- or down-regulated in malignancies, giving them an oncogenic or tumour-suppressor effect. MiRNA expression has been linked to clinical outcomes and tumour regulation. Identification of tumour-specific miRNA signatures may assist the discovery of new biomarkers with diagnostic and prognostic utility. Here, we aimed to detect the expression of different miRNAs in different paediatric brain tumour subtypes to discover biomarkers for early detection and develop novel therapies. Expression of 82 miRNAs was detected in 120 paediatric brain tumours from fixed-formalin paraffin-embedded (FFPE) tissues, low-grade glioma, high-grade glioma, ependymoma, and medulloblastoma, using quantitative real-time PCR (qRTPCR). qRT-PCR analysis showed significant differences in miRNA expression between tumour subtypes (P<0.05). Low expression of miR-221, miR-9, and miR-181c/d and over-expression in miR-101, miR-222, miR-139, miR-1827, and miR-34c was found in medulloblastoma; low expression of miR-10a and over-expression of miR-10b and miR-29a in ependymoma; low expression of miR-26a and overexpression of miR-19a/b, miR-24, miR-27a, miR- 584, and miR-527 in low-grade glioma. Multivariate Cox regression showed specific miRNA expression between responders and non-responders. The most specific were miR-10a and miR-29a low expression in LGG non-responders, miR-135a and miR-146b over-expression in ependymoma non-responders, and miR-135b overexpression in medulloblastoma non-responders. MicroRNAs are differentially expressed between subtypes of paediatric brain tumours suggesting that they may have a significant role in diagnosis. A greater understanding of aberrant miRNA expression in paediatric brain tumours may support development of novel therapies. Characterization of tumour-specific miRNA signatures may have an important role in the discovery of biomarkers with diagnostic or prognostic utility.

Poster abstracts


78

38

Creating a suitable model for TONSL-mediated oncotherapy

Matthew A.M. Todd1, Anja Groth1

1 Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, DENMARK

Genotoxic insults from endogenous and exogenous sources including oncogenic signaling can lead to replication fork stalling and collapse, jeopardizing the process of DNA replication and ultimately genome integrity. Cells have evolved an elaborate network of replication fork protection and DNA repair mechanisms to prevent replication-associated DNA damage. Cancer cells are particularly dependent upon DNA repair machinery to cope with high loads of replication stress that accompany their loss of checkpoints and accelerated proliferation. Homologous recombination (HR) offers the highest fidelity for DSB repair, providing a potentially valuable cancer target. We and others have defined a role for TONSL-MMS22L in assembling post-replicative chromatin and HR [1-6]. In cooperation with collaborators, our group previously described the structural basis for the interaction between the TONSL ARD domain and H4K20me0, which marks newly synthesized H3-H4 [2]. This interaction provides a mechanism for the recruitment of TONSL to post-replicative chromatin, an environment in which sister chromatids are available for HR. Intriguingly, histone-binding mutations of TONSL were toxic when expressed in a cancer cell model [2], suggesting that disruption of the TONSL-H4K20me0 interaction could be applied in a therapeutic context. Here, we present our strategy to further investigate the cellular mechanisms reliant upon TONSL-mediated HR with the aim of identifying candidates for synthetic lethality in human cancers. TONSL will be co-depleted against an siRNA library and using a reporter model, we will measure cell viability and DNA damage by high content microscopy. It is anticipated that the outcome of this screen will delineate which pre-clinical models are most suited to therapies targeting TONSL.

1. Duro, et al. 2010. PMID: 21055984.

2. Saredi, et al. 2016. PMID: 27338793.

3. O’Donnell, et al. 2010. PMID: 21055983.

4. O’Connell, et al. 2010. PMID: 21055985.

5. Piwko, et al. 2010. PMID: 21113133.

6. Piwko, et al. 2016. PMID: 27797818.

Poster abstracts


79

39

FOXO maintains genomic stability by regulating histone acetylation around double-strand breaks

Sabina van Doeselaar1, Miranda van Triest1, Boudewijn Burgering1

1 University Medical Center Utrecht, Utrecht, NETHERLANDS

Members of the Forkhead Box O (FOXO) family are transcription factors downstream of the PI3K-PKB/AKT signaling pathway. They regulate cellular processes involved in cancer an ageing, such as cell death, metabolism, cell cycle, survival and growth. Besides these functions, they are also known to have a role in DNA damage repair via the regulation of transcription of two DNA damage repair protein, namely GADD45 and DDB1. We show that FOXOs also have a direct role in DNA repair via localization to the DNA break. FOXO localizes to double strand breaks in an ATM-dependent manner via association with the MRN complex. FOXO does this in complex with the histone acetyltransferase Tip60 and the adaptor protein TRRAP. Tip60 induces the localized acetylation of lysine 16 on histone 4 (H4K16), which results in several downstream effects. First, this results in the removal of 53BP1 off the DNA and this promotes DNA repair via homologous recombination over error-prone non-homologous end-joining. Second, we show that chromatin remodeling complex members TBL1X (NCOR complex) and p66 (NURD complex) have a lower binding affinity for acetylated than for non-acetylated H4K16. This may indicate that chromatin remodeling complexes are removed from the chromatin near double strand breaks, perhaps to make room for DNA repair protein. Overall, our findings show a new and direct function of FOXOs in the DNA damage response.

Poster abstracts


80

40

Reciprocal regulatory links between transcription and DNA double strand break repair

Alexandra Vitor1, Robert Martin1, Sreerama Sridhara1, João Sabino1, Ana Rita Grosso1, Sérgio de Almeida1

1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, PORTUGAL

A major question in molecular biology refers to the crosstalk between different cellular processes that act within the same chromatin space. This gains particular relevance if we are referring to transcription and DNA repair, both inducing a considerable chromatin remodeling. Histone modifications are important mediators of the interplay between the DNA damage response and transcription. Typically, actively transcribed genes are “marked” by a characteristic set of histone modifications. One of the chromatin marks of transcription is the trimethylation of histone H3 (H3K36me3) by the SETD2 methyltransferase. Previous work in our lab and others has shown that the presence of H3K36me3 in coding regions promotes the repair of DNA double strand breaks (DSBs) by the error-free homologous recombination pathway, in order to safeguard the integrity of genetic information. The question that follows is: How do DNA DSBs impact on transcription? Currently in the field there is the lack of experimental model systems to simultaneously follow transcription and DNA repair with single-molecule sensitivity, in live cells. Therefore, to answer this question and to gain detailed insights with unprecedented resolution on such events, we established a new in vitro model system that makes use of an endonuclease-triggered DSB within an inducible gene to follow, in real-time, transcription upon the induction of a single DNA DSB, by using spinning disk confocal microscopy. After the establishment of the system, we confirmed that a proper damage signaling response was being activated. Therefore, with this tool we are currently unraveling the molecular details that take place immediately after the transcription machinery encounters a DNA DSB.

Poster abstracts


81

41

MCM2 function(s) in histone chaperoning and guarding cell identity

Alice Wenger1, Caroline Bianchi Strømme1, Anja Groth1

1 Biotech Research & Innovation Centre (BRIC), Copenhagen, DENMARK

The packaging of DNA with histones into higher-order chromatin structures is crucial for genome stability and contributes to genome function. DNA replication poses a genome-wide disruption of chromatin organization as both genomic and epigenomic information need to be duplicated with high fidelity to maintain genome integrity and function in daughter cells. In proliferating cells, histones are evicted in front of replication forks and recycled onto daughter DNA strands in coordination with new histone deposition. Post-translational modifications of recycled histones are thought to be instructive for the modification of new ones, and are thus of major importance for restoration of chromatin structure and cellular memory. However, the molecular processes of histone recycling remain unclear.

We and others identified MCM2 (minichromosome maintenance complex component 2) – a core component of the replicative helicase – as key candidate for histone recycling at the replication fork. Based on our crystal structure of MCM2 chaperoning histones H3/H4, we are introducing point mutations in the histone-binding domain at the endogenous MCM2 locus in mouse ES cells using genome editing technologies. The aim of this project is to test the role of MCM2 in histone recycling during replication and cell fate decision.

1. Groth A, Corpet A, Cook AJ, Roche D, Bartek J, Lukas J, Almouzni G. (2007). Regulation of replication fork progression through histone supply and demand. Science 318(5858):1928-31.

2. Foltman M, Evrin C, De Piccoli G, Jones RC, Edmondson RD, Katou Y, Nakato R, Shirahige K, Labib K. (2013). Eukaryotic replisome components cooperate to process histones during chromosome replication. Cell Rep. 3(3):892-904.

3. Huang H, Strømme CB, Saredi G, Hoedl M, Strandsby A, González-Aguilera C, Chen S, Groth A, Patel DJ. (2015). A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks. Nat Struct Mol Biol. 22(8):618-26.

Poster abstracts


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Standardization of the pre - analytical stage of isolation of circulating microRNA

Ilgar Guseinov1, Alexander Abramov1, Alisa Petkevich1, Dmitrii Chebanov1, Pavel Ogurtsov1, Liya Kanakhina1

1 Peoples’ Friendship University of Russia (RUDN University), Moscow, RUSSIAN FEDERATION

MiRNA maybe a promising prognostic and predictive biomarker of difference biological conditions. Along with specificity and sensitivity problems of these biomarkers, there is a problem of reproducible recovery of these molecules from biological samples, which is especially important for implementation into routine clinical practice. In this study, we took blood samples from 8 healthy donors and formed 6 groups for each sample depending on different conditions (tube type, time between obtaining material and extraction, freezing samples).

According to our data, following trends were found out: samples with highest concentrations with miRNA (21, 145, and 16) were those centrifugated and isolated during 60 min after obtaining biological sample (blood) from donors. Surprisingly, samples following recovery after freezing (-20◦C) were with higher miRNA concentration then those which were being exposed for 48 hours at room temperature (+24◦C).

Moreover, there is a significant difference between tube types (Streck RNA Tube 10 ml and S-Monovette® 2.6 ml, K3E EDTA). We suppose this result can be explained by different interaction of tube substances and substances of isolation kits, also this difference may be due to variable stability of samples in different tubes.

Further research in needed for creation standardized protocol for reproducible recovery of miRNA from blood samples. So, our next step is to study intracellular and extracellular miRNA concentrations and assess hemolysis.

Poster abstracts


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44

Research of microRNA level in blood plasma of patients with melanoma

Liya Kanakhina1, Alexandr Abramov1, Ilgar Guseinov1, Pavel Ogurtsov1, Dmitriy Chebanov1

1 RUDN University, Moscow, RUSSIAN FEDERATION

The number of miRNA depends on type of disease and the effectiveness of the treatment provided. The presence of a number of miRNAs in plasma makes them as potential indicators for disease monitoring, as well as its prognosis. Based on published research analysis, we have focused our work on the following specific for melanoma ten microRNAs, but the results of our study show significant differences at the levels of let-7a, miRNA-106a, miRNA-199a and miRNA-221. Our research work aimed at isolating microRNA from the cancer plasma, assessing level of microRNAs and exosomal microRNAs, and reviewing and comparing microRNA amount among various groups: of patients with melanoma, with metastases, with stable remission and healthy donors. To conduct research a real-time PCR with primers for specific microRNAs has been used. Patients without metastases had the least amount of miRNAs: 1 898 144,85 copies / μl, 3 951 463,94 copies / μl, 2 029 141,03 copies / μl, 2 660 184,96 copies / μl, respectively. Patients with remission seemed to have maximum miRNAs number: 2 381 799,79 copies / μl, 3 903 427,21 copies / μl, 4 244 876,11 copies / μl, 13 073 481,78 copies / μl, respectively. In terms of exosomal microRNAs our research reveals the following findings: the longer a patient has had melanoma, the bigger amount of miRNA-199a and miRNA- 221 has been found. The number of let-7a and miRNA-106a exosomal miRNAs in patients with melanoma and with remission shown to be much smaller than amount of miRNA-199a and miRNA-221 in the same patient categories accordingly. In the healthy patientsthe number of copies of let-7a miRNA and miRNA-106a is 4 and 7 times greater than the average number in melanoma patients. We believe that the results of our research can be used to evaluate the effectiveness of a treatment provided.

Poster abstracts


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45

Mechanisms at work that enhance chromatin dynamics and recombination rates in response to double-strand breaks and oxidative damage

Anaïs Cheblal1, Michael H. Hauer1, Andrew Seeber1, Assaf Amitai2, Susan M. Gasser1

1 Friedrich Miescher Institute for Biomedical Research, Basel, SWITZERLAND, 2 MIT, Cambridge, MA, USAChromatin is in constant motion within the nucleus. Changes in DNA dynamics upon DNA damage have been extensively studied by fluorescence microscopy and mean squared displacement analysis, leading to diametrically opposed proposals for the forces behind this movement. We have exploited an analytical workflow based on an improved imaging regime with higher spatial and temporal resolution and statistical analyses that extract biophysical parameters from the trajectories. We have applied this to time-lapse imaging of an inducible double-strand break in yeast. Based on the extracted parameters our modeling predicts chromatin expansion near a break, which we confirm by super-resolution microscopy. We are able to differentiate between extrinsic forces arising from the cytoskeleton and intrinsic forces stemming from changes in local chromatin structure. The actin cytoskeleton contributes to the former, while the INO80 chromatin remodeler is required for local chromatin expansion. We do not rule out that loss of an extrinsic tether, such as centromere detachment, could occur and even alter chromatin dynamics, as reported by others (Strecker et al., 2016). However, our data makes it highly unlikely to be the only driving force behind break movement. Rather, we argue that the expansion of chromatin at the site of the break and the local reorganization of chromatin that subsequently occurs drives increased movement. Using a range of quantitative methods, we show that histone levels drop by 20-40% in response to DNA damage, due to eviction from chromatin by the INO80 remodeler and degradation by the proteasome. This eviction is likely responsible for the observed chromatin decompaction and increased fiber flexibility. This leads to enhanced recombination rates and DNA repair focus turnover. Thus, we propose that a generalized reduction in nucleosome occupancy is an integral part of the DNA damage response, providing mechanisms for enhanced chromatin mobility and homology search.

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