Copyright © 2019 Single-molecule imaging of transcription ... · (PROP) region downstream the transcription start site and a region within an internal exon (EX2). Induction of a

SC I ENCE ADVANCES | R E S EARCH ART I C L E

GENET I CS

Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina daUniversidade de Lisboa, Lisboa, Portugal.*Present address: UCIBIO-REQUIMTE, Departamento de Ciências da Vida,Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516Caparica, Portugal.†Corresponding author. Email: [email protected] (R.M.M.);[email protected] (S.F.d.A.)

Vítor et al., Sci. Adv. 2019;5 : eaau1249 9 January 2019

Copyright © 2019

The Authors, some

rights reserved;

exclusive licensee

American Association

for the Advancement

of Science. No claim to

originalU.S. Government

Works. Distributed

under a Creative

Commons Attribution

NonCommercial

License 4.0 (CC BY-NC).

Dow

nloade

Single-molecule imaging of transcriptionat damaged chromatinAlexandra C. Vítor, Sreerama C. Sridhara, João C. Sabino, Ana I. Afonso, Ana R. Grosso*,Robert M. Martin†, Sérgio F. de Almeida†

How DNA double-strand breaks (DSBs) affect ongoing transcription remains elusive due to the lack of single-molecule resolution tools directly measuring transcription dynamics upon DNA damage. Here, we establishednew reporter systems that allow the visualization of individual nascent RNAs with high temporal and spatial res-olution upon the controlled induction of a single DSB at two distinct chromatin locations: a promoter-proximal(PROP) region downstream the transcription start site and a region within an internal exon (EX2). Induction of aDSB resulted in a rapid suppression of preexisting transcription initiation regardless of the genomic location.However, while transcription was irreversibly suppressed upon a PROP DSB, damage at the EX2 region drovethe formation of promoter-like nucleosome-depleted regions and transcription recovery. Two-color labeling oftranscripts at sequences flanking the EX2 lesion revealed bidirectional break-induced transcription initiation.Transcriptome analysis further showed pervasive bidirectional transcription at endogenous intragenic DSBs.Our data provide a novel framework for interpreting the reciprocal interactions between transcription and DNAdamage at distinct chromatin regions.

d fr
on F
ebruary 27, 2020http://advances.sciencem

ag.org/om
INTRODUCTIONDNA is the template for vital cellular processes such as replication,transcription, and DNA repair. Thus, it is expected that sometimes,two of these processes simultaneously use the exact same sequenceand collide. Such conflicts may have catastrophic consequences andare an important source of aberrant gene expression programs andgenome instability (1, 2). Intriguingly, recent studies revealed thatclose encounters between transcription and the DNA repair machineriesmay have important physiological outcomes. For instance, complexesinvolved in scheduled DNA damage have been shown to coactivatetranscription (3–6), and transcription at double-strand breaks (DSBs)has been suggested to play an essential role in DNA repair (7). Thesefindings hint to physiologically productive reciprocal interactions be-tweenDNA-templated processes that are still greatly unappreciated (1).
High-resolution single-molecule imaging in live cells has been usedto dissect distinct gene expression parameters, such as kinetics of RNApolymerase II (RNAPII) elongation during transcription (8). However,despite growing interest in the interplay between gene expressionandDNAdamage, the impact of a DSB on transcription has not beeninspected with the temporal and spatial resolution that single-moleculeimaging tools could allow. Here, we sought to obtain rigorous mea-surements of transcription dynamics within the first minutes after in-duction of a single DSB at distinct chromatin regions. Using novelreporter genes, we reveal that rapid suppression of preexisting transcrip-tion initiation is a common feature induced by DSBs. In addition,we observed that transcription recovery uponDNAdamage is restrictedto intragenic chromatin regions and relates to the formation of anucleosome-depleted region. Our data provide a clear view of theimpact of DSB on elongating RNAPII complexes and disclose hithertounappreciated patterns and kinetics of break-induced transcription.

RESULTSEngineering reporter genes to image single-moleculetranscription upon controlled induction of a DSBWe used human embryonic kidney (HEK) 293 cells containing a singleFlp recombinase target (FRT) genomic locus for Flp-in homologous re-combination of a single copy of a modified immunoglobulin M (IgM)gene driven by a doxycycline-inducible cytomegalovirus (CMV)promoter (Fig. 1A). Two independent reporter genes were generatedupon insertion at distinct regions of a unique I–SceI restriction site,which is absent from the human genome: insertion 61 base pairs (bp)downstream the transcription start site at a promoter-proximal region(PROP reporter; Fig. 1B and fig. S1A) and insertion inside exon 2 (EX2reporter; Fig. 1B and fig. S1B). To image transcription in real time usingthe PROP reporter, we inserted 24 repeats ofMS2 sequences immediatelydownstream the I–SceI site. In the EX2 reporter, we flanked the I–SceIsite with 24 repeats of MS2 and PP7 phage sequences. Transcription isvisualized through the recruitment of fluorescently tagged MS2- andPP7-binding proteins to single nascent transcripts (Fig. 1, C and F)(8). The two-color labeling strategy designed for the EX2 reporter allowsfor interrogation of transcription at both regions flanking the DSB. Inintact (i.e., without DSB induction) EX2 reporter cells, analysis oftranscription revealed a strong correlation between the MS2 and PP7fluorescent signals throughout the entire imaging period (Fig. 1F).The two-color labeling had a sufficient resolution to capture the temporaldelay between transcription of theMS2 sequences and transcription ofthe PP7 sequences located downstream (Fig. 1F and fig. S2A). More-over, we could detect the increase in fluorescence intensity correspondingto the sequential binding of the MS2-RFP (red fluorescent protein) andPP7-GFP (green fluorescent protein) proteins to their target sequenceson single RNA transcripts as they emerge fromRNAPII (fig. S2, B andC).By computing the MS2 or PP7 sequence length with the time intervalsof differentmetrics between initial detection of fluorescence values and/or maximum values, we determined the average transcription ratesranging from 3.8 to 4.9 kb/min at the MS2 and PP7 sequences, respec-tively (fig. S2D). These values, which are in agreement with the approx-imate rates of 4 kb/min estimated in previous studies (9, 10), reveal thatthe reporter genes are transcribed normally.

1 of 12

http://advances.sciencemag.org/


on February 27, 2020


Dow

nloaded from

A

C

B

E

F

CMV promoter 2tetO Reporter gene

Single genomic integration

MS2SL PP7SL

I–SceI

MS2SL

I–SceI

pCMV

2tetO

Exon I

Exon I

Intron

Intron

Exon II

Exon II

PROP

EX2

pCMV

2tetO

3 min

4

3

TS

n0 min

21

TS

n

3 4TS21TSMS2-GFP

21 43TSTS

1

2

0 min

TS

n

1 min

3

4TS

n

MS2-RFP 5 µmPP7-GFP

100 120 140 160 180 200 220 240 260 280 300806040200Time (s)

1

Time (s)0 12 24 36 48 60

PROP

n tra

nscr

ipts

1

2

3 n tra

nscr

ipts

100 120 140 160 180 200 220 240 260 280 300Time (s)

806040200

EX2

n tra

nscr

ipts

1

2

3

1

n tra

nscr

ipts

Time (s)0 12 24 36 48 60

pCMV

2tetOMS2SL

Pol II Pol II Pol II

MS2-GFP

mRNA

pA

PROP D

MS2SL PP7SLpCMV

2tetO

Pol II Pol II Pol II

PP7-GFPMS2-RFP

mRNA

pA

EX2

Fig. 1. New reporter genes allow visualization of transcription upon DNA damage with single-molecule resolution. (A) Illustration of a HEK293 cell containing asingle FRT genomic locus for single copy integration of the reporter gene. (B) Schematic of the two reporter genes with the I–SceI site and MS2/PP7 sequenceshighlighted. (C and D) Representative images of PROP (C) and EX2 (D) reporter cells. Images were acquired at the indicated time points after doxycycline treatment.Examples of pre-mRNAs diffusing throughout the nucleoplasm are numbered 1 to 4. The transcription site (TS) of the reporter gene is indicated in each cell.Corresponding images depicting the diffraction limited objects in the highest intensity plane are shown below. (E and F) Time-lapse series of imaged PROP (E)and EX2 (F) reporter cells upon doxycycline treatment. The number (n) of transcripts was plotted over time in line graphs. Enlarged line plots depict a complete cycleof fluorescence gain and loss for each of the time-lapse series.

Vítor et al., Sci. Adv. 2019;5 : eaau1249 9 January 2019 2 of 12





Dow

nloaded from

A

B

Transienttransfection +TA

I–SceI–GR-iRFP

MS

2-R

FPM

DC

1-G

FP

1 min 7.5 min 10 min 16 min

5 µm12 16

bg

max

Nor

m.

Mea

n FI

(a.u

.)

Time (min)840

MDC1-GFPMS2-RFP

0

4

8

12

16

20

n tra

nscr

ipts

0Time (min)

10 20 30 40 50 60

0

4

8

12

16

20

n tra

nscr

ipts

0Time (min)

10 20 30 40 50 60

C

D

MS2-GFP

No TA

+TA

0 min 30 min 50 min15 min 45 min


0

20

40

60

80

100

%

Term. Contin.

n = 11n = 2No TA

0

20

40

60

80

100

%

Term. Contin.

n = 3n = 20+TA

0 min

No TA

8 min

+TA

3 min

+TA

iRFP

I–SceI

GR

Fig. 2. PROP DSBs silence transcription. (A) PROP reporter cells were transfected with the I–SceI–GR-RFP fusion protein and imaged for 8 min after TA treatment.Images acquired at the indicated time points illustrating the translocation of the enzyme into the nucleus are shown. (B) Dynamics of transcription revealed by MS2-RFPfluorescence intensity at the transcription site and of MDC1-GFP recruitment to the transcription site in PROP cells transfected with I–SceI–GR fusion proteins and live-imaged after TA treatment. Representative images acquired at the indicated time points are shown. The dual-line plot on the right-hand side represents the averagelabeling intensity of MDC1-GFP and MS2-RFP quantified for a region of interest (ROI) defined around the transcription site over time. One representative experimentfrom more than 30 individual cells recorded is shown. a.u., arbitrary units. (C and D) Time-lapse, multiplane, spinning disk confocal microscopy was used to monitor livecell transcription dynamics of PROP reporter cells expressing MS2-GFP in the absence of (C) or upon (D) TA treatment. Representative line plots of number (n) oftranscripts over time are shown. Bar graphs on the right-hand side represent the frequency of cells that exhibit a pattern of continuous (Contin.) or suppressed/terminated (Term.) transcription revealed by the lack of MS2-GFP fluorescence. The absolute number (n) of imaged cells is shown above each bar.




Do

Regulated induction of a single DSB at distinct genic regionsTo control the timing ofDSB induction,we transfected the reporter cellswith an I–SceI enzyme fused to the glucocorticoid receptor (I–SceI–GR).This fusion was fully able to digest the reporter genes in vitro (fig. S3A).Within 3 min after cell treatment with triamcinolone acetonide (TA),cytoplasmic I–SceI–GR translocated into the nucleus (Fig. 2A). Inthe nucleus, I–SceI–GR has access to its restriction site on each reportergene to induce a single genomic DSB. To confirm this, we measuredthe dynamics of mediator of DNA damage checkpoint 1 (MDC1), afactor that plays an early role in the DNA damage response (DDR)(11, 12), through live cell microscopy imaging of PROP (Fig. 2B) andEX2 (fig. S3B) reporter cells. Approximately 12 min after the additionof TA, we observed MDC1 recruitment to the transcription site,confirming that I–SceI translocation into the nucleus cut the reportergenes. In parallel to MDC1 recruitment, we also observed a strong re-duction of the transcription levels of both reporter genes (Fig. 2B andfig. S3B), suggesting that aDSB leads to rapid suppression of preexistingtranscription initiation.


The impact of a DSB on transcription depends on its locationwithin the geneRecent studies reported break-induced transcription at the DNA endsof a DSB (3–6). While suppression of the canonical preexisting tran-scription initiation was the immediate response to a DSB regardlessof its location within the reporter gene, we sought evidence of break-induced transcription initiation upon a DSB. In our reporters, the PP7and/or MS2 sequences were inserted in close proximity to the I–SceIsite. This implies that any transcription event at the DSB should bereadily detected by the recruitment of fluorescent PP7- and/or MS2-binding proteins. To address this hypothesis, we first imaged nondam-aged PROP reporter cells for 50 min. Under these conditions, wecontinuously detected transcription of the reporter gene, yielding anumber of nascent RNA transcripts that oscillated between 1 and 18(see Materials and Methods and figs. S4 and S5 for details on the es-timation of the transcript number) (Fig. 2C). As expected, induction ofthe PROP DSB resulted in a rapid suppression of full-length tran-scription of the reporter gene within approximately 10 min after the



wnloaded from

MS2-RFP PP7-GFP

No TAn = 22n = 3

Term. Contin.0

20

40

60

80

100

%

0

2

4

6

8

10

n tra

nscr

ipts

0Time (min)

10 20 30 40 50 60

A0 min 30 min 60 min15 min 45 min

B

n = 3n = 13

Resume Term.0

20

40

60

80

100

%

+TA

0

2

4

6

8

10

n tra

nscr

ipts

0Time (min)

10 20 30 40 50 60


+TA

0

2

4

6

8

10

n tra

nscr

ipts

0Time (min)

10 20 30 40 50 60

C


+TA

No TA

D

DNA ligase IV RNAi

α-Tubulin

Total histone H3

ctrl kd

Resume Term.

n = 10n = 8

0

20

40

60

80

100

%

DNA ligase IV RNAi+TA

n = 6n = 7

Coupl. Uncoupl.0

20

40

60

80

100

%

+TA

Fig. 3. Break-induced transcription initiation. Time-lapse, multiplane, spinning disk confocal microscopy recordings of EX2 reporter transcription dynamics in livecells revealed by the dual labeling of nascent transcripts with MS2-RFP and PP7-GFP in the absence [(A) no TA] or upon [(B and C) +TA] DNA damage. The dual-lineplots represent the number (n) of transcripts detected at the transcription site over time. The bar graph in (A) shows the frequency of cells that exhibit a pattern ofcontinuous (Contin.) and suppressed/terminated (Term.) transcription. The bar graph in (B) shows the frequency of cells, where preexisting transcription was sup-pressed in response to TA treatment and either resumes (Resume) or remains silenced (Term.) during the entire imaging period. A representative pattern oftranscription resuming is depicted on the images and dual-line plot in (B). (C) Representative images and dual-line plot of a recorded cell where only the PP7-GFPfluorescence is observed after the damage-induced termination and before dual-labeled transcripts are detected. The bar graph depicts the frequency of cells thatexhibit a similar uncoupling of the MS2-RFP and PP7-GFP fluorescent signal (Uncoupl.) and those where transcription resumed as illustrated in (B) (Coupl.). (D) Westernblot analysis of DNA ligase IV in control (ctrl) or DNA ligase IV small interfering RNA (siRNA)–depleted cells. a-Tubulin and total histone H3 serve as loading controls. Thebar graph depicts the frequency of cells that either resume or irreversibly terminate transcription after induction of the DSB in DNA ligase IV–depleted cells. kd,knockdown; RNAi, RNA interference.

4 of 12





Dow

nloaded from

MS2SL

I–SceI

pCMV

2tetO

Exon IIntron

Exon II

MS2SL PP7SL

I–SceIExon IIntron

Exon IIpCMV

2tetO

A

B D

C

Fig. 4. Break-induced chromatin modifications. (A) Nucleosome occupancy measured by total histone H3 ChIP at two regions (marked A and B in the schematics ofthe reporter gene) flanking the I–SceI site of the EX2 reporter. (B) Levels of the indicated histone modifications at the A and B regions of the EX2 reporter genemeasured by ChIP before and after (30 min and 1 hour) the addition of TA to induce a DSB. (C) Nucleosome occupancy and (D) levels of histone modifications ata region (marked C) immediately downstream the I–SceI site of the PROP reporter gene. All data were normalized against the nucleosome occupancy on non–TA-treated cells and represent mean and SEM from at least three independent experiments. *P < 0.05 and **P < 0.01, obtained using two-tailed Student’s t test.




Dow

nload

addition of TA in 87% of imaged cells (Fig. 2D). Transcription of thePROP reporter gene remained silenced throughout the remainingimaging period, as revealed by the lack of anyMS2-GFP fluorescencerecovery (Fig. 2D). We conclude that the PROPDSB suppresses full-length transcription of the reporter gene and that this is irreversibleon the time scale of the experiment.

In EX2 reporter cells, canonical transcription was continuouslyobserved in the absence of DNA damage, as revealed by the overlapbetween MS2-RFP and PP7-GFP fluorescence signals (Fig. 3A). UponTA treatment, induction of the DSB led to a complete suppression ofpreexisting transcription initiation detected by the loss of both MS2-RFP and PP7-GFP signals at the transcription site (Fig. 3B). Increasingthe temporal resolution to 10-s imaging intervals further showed that,after a DSB, RNAPII elongates until the break site, as revealed by thedetectionofMS2-RFP fluorescence alone, before a complete transcriptionshutdown is observed (fig. S3C). However, in contrast to the PROPreporter, full-length transcription of the damaged EX2 reporter generecovered in the majority (81%) of imaged cells (Fig. 3B). Strikingly,the pattern of transcription recovery was not unique. In 54% of thecells, we detected a late transcription recovery, approximately 30 to


40 min after DSB induction, whereas in the remaining cells, tran-scription recovered immediately after suppression of the preexistingtranscription (Fig. 3C). Moreover, the late transcription recovery wassignificantly (P < 0.05) suppressed in DNA ligase IV–depleted cells(Fig. 3D), which are incompetent for nonhomologous end joining (13).This suggests that DNA repair is necessary before RNAPII resumeselongation across the damage site. The early transcription recoverywas detected by the accumulation of PP7-GFPbut not ofMS2-RFPpro-teins (Fig. 3C), indicating that transcription was initiated between theMS2 and PP7 sequences, consistently with break-induced transcriptioninitiation from the DSB.

Locus-specific changes to the chromatin landscapesurrounding DSBsThe finding that recovery of transcription is greatly determined by thelesion site highlights chromatin structure as an important determinantof this outcome. Previous studies revealed that changes in chromatinstructure, such as local nucleosome depletion, are sufficient to drivetranscription initiation from noncanonical promoter sequences (14, 15).With this in mind, we sought evidence of break-induced changes in



ed from

B

0

1

2

3

4

5

n tra

nscr

ipts

0Time (min)

10 20 30 40 50 60

MS2-RFP (antisense)PP7-GFP (sense)

+TA

020406080

100

%

No AStranscr.

AStranscr.

30 min0 min 15 min 53 min45 min

A

PP7SL

I–SceIExon I Intron Exon II

pApCMV 2tetO

MS2SL

5′

5′Pol II

PP7-GFP

3′

3′

PP7SL

I–SceIExon I Intron Exon IIpApCMV 2tetO

MS2SL

5′

5′

MS2-RFP

Pol II3′

3′

Fig. 5. Intragenic DNA damage drives bidirectional break-induced transcription initiation. (A) Schematics of the EX2-AS reporter constructed to directly inspectbreak-induced antisense transcription initiation. (B) Representative images of a total of eight imaged cells and dual-line plot of a recorded cell where break-inducedantisense transcription initiation was detected. The bar graph depicts the frequency of cells where antisense transcription was either not observed (No AS transcr., 50%)or observed (AS transcr., 50%).

6 of 12



http://advD

ownloaded from

nucleosome occupancy at the vicinity of the DSB. Histone H3 chro-matin immunoprecipitation (ChIP) assays before and after induction ofthe DSB revealed a significant loss of nucleosomes at the chromatinregion near the I–SceI site of the EX2 reporter (Fig. 4A). Nucleosomedepletion was further confirmed by micrococcal nuclease (MNase)digestion assays (fig. S6). In agreement with the finding that tran-scription was not suppressed following the intragenic DSB, we didnot observe a reduction in the relative levels of histone modificationsthat mark active chromatin, H3K9Ac and H3K36me3 (Fig. 4B). Weobserved slightly increased levels of H3K9Ac, a histone modificationthat decorates active promoter regions, at the DSB site (Fig. 4B),which may suggest break-induced tailoring of a promoter-like chro-matin landscape and is compatible with the observed break-inducedtranscription initiation.

Induction of a DSB at the I–SceI site of the PROP reporter gene didnot cause nucleosome depletion (Fig. 4C). Moreover, the levels ofH3K36me3 decreased significantly within 30 min after the DSB, andno changes were detected in the levels of H3K9Ac (Fig. 4D). These dataare consistent with the irreversible suppression of transcription ob-served upon induction of a PROP DSB.

Intragenic DSBs induce bidirectional transcription initiationNucleosomes play an important role in restricting antisense tran-scription (16, 17). The finding that intragenic DSBs reduce nucleosomeoccupancy raises the hypothesis that transcriptionmay initiate from thebroken DNA ends in the sense and antisense direction. To further test


the possibility of bidirectional transcription in response to a DSB, weimaged transcription of a new reporter gene obtained by invertingthe 24 MS2 repeat tract of the EX2 reporter (Fig. 5A and figs. S1Cand S3D). In the resulting EX2-antisense (EX2-AS) reporter, recruit-ment of MS2-RFP proteins to the nascent RNA transcript occurs onlyif the MS2 sequences are transcribed in the antisense direction. OnlyPP7-GFP proteins were detected at the site of transcription of theEX2-AS reporter gene in the absence of DNA damage (Fig. 5B). UponTA treatment, the I–SceI–induced DSB suppressed preexisting sensetranscription (revealed by the loss of PP7-GFP) and drove antisensetranscription initiation in 50% of cells, as shown by the accumulationof MS2-RFP fluorescence at the site of transcription (Fig. 5B).

Pervasive break-induced transcription initiation atendogenous DSBsTo gain further insight into bidirectional break-induced transcriptioninitiation, we analyzed DSBCapture and transcriptome data obtainedfrom normal human epidermal keratinocytes (NHEKs) (18). Analysisof DSBCapture data provided a genome-wide profile of endogenousDSBs at a single-nucleotide resolution. From these, we selected thosethat map within the body of active genes and interrogated transcrip-tome data for antisense transcriptionwithin 1 kb from theDSB (Fig. 6A).This analysis revealed amarkedpeak immediately upstream theDSB sites(Fig. 6A). Permutation analysis further revealed statistically significantlevels of antisense transcription at the intragenic DSB loci when com-pared to random genic regions (Fig. 6B). In agreement with the data


ances.sciencemag.org/

DSBs

3.9

13.8

23.7

–1 kb DSB 1 kb

−2 Z score 5

DNase

2.5

13.1

23.8

–1 kb DSB 1 kb

−2 Z score 7

Antisense

0.2

0.4

0.7

–1 kb DSB 1 kb

−2 Z score 10

RP

KM

A B

Fig. 6. Pervasive antisense transcription at endogenousDSBs. (A) Metaprofiles and heatmaps of DSBs, DNase accessible regions, and antisense transcription centeredon the position of intragenic DSBs. (B) Fold enrichment of antisense transcription upstream the DSB calculated as the number of transcribed DSB regions divided by thenumber of randomly shuffled DSBs overlapping transcribed regions. Error bars depict SEM of fold enrichment over random. Permutation analysis, ***P < 0.001.

7 of 12



obtained with our reporter gene, DSB and antisense transcription peaksoverlapped with nucleosome-depleted regions [deoxyribonucleasesequencing (DNase-seq)], indicating an association between regulatorychromatin andantisense break-induced transcription (Fig. 6A).Together,these data suggest that bidirectional transcription initiation is awidespreadfeature of intragenic DSBs that is related with the formation of break-induced nucleosome-depleted regions.



Dow

nloaded from

DISCUSSIONHere, we imaged single nascent RNAs at the site of transcription togather quantitative information about the dynamics of transcriptionat damaged chromatin. We provide accurate kinetic parameters ofthe timing of transcription suppression upon a DSB, complementingprevious reports that correlated DNA damage with repression oftranscription (19–25). Moreover, we demonstrate that transcriptionrecovery after break-induced suppression depends on the location ofthe DSB.While a lesion in the beginning of the transcription unit at apromoter proximal region prevented recovery during the entire imag-ing period, DNA damage within the gene body permitted transcriptionrecovery with seemingly distinct kinetic patterns: late (i.e., 30 to 40 minafter induction of a DSB) and early (immediately after suppression ofthe preexisting transcription initiation). The two-color labeling of thenascent RNAs revealed that RNAPII elongated through the I–SceI siteduring the late recovery, suggesting that the broken DNA ends hadalready been resealed. In agreement, preventing DNA repair throughdepletion of DNA ligase IV significantly decreased the number of cellswhere transcription recovery was observed. In contrast, the earlytranscription recovery is not consistent with the kinetics of DNA repair,which requires protein effectors that are recruited to the DSB severalminutes after the lesion (26). Instead, our data suggest that brokenDNA ends are competent to drive bidirectional transcription initiationboth in our reporter genes and widespread across the human genome.This is supported by the finding that an intragenic DSB nucleates apromoter-like chromatin landscape characterized by nucleosome de-pletion. Our data further suggest that each DSB promotes transcriptioninitiation in one direction only, with no apparent preference for eithersense or antisense. This is illustrated by the failure to simultaneouslyobserve PP7- and MS2-containing transcripts at the Ex2-AS reporter.This notion is consistent with the detection of break-induced tran-scription initiation at the EX2 reporter (which does not allow visualiza-tion of antisense transcription) in only approximately half of the cells.These data raise the hypothesis that, at each DSB, break-induced tran-scription initiation occurs exclusively in a single direction, and suggestthe existence of a mechanism preventing RNAPII firing in the oppositedirection. The observed 50% frequency of antisense transcription sug-gests that the direction of break-induced transcription is decided in astochastic manner, perhaps determined by the kinetics of RNAPIIbinding to one of the two broken DNA ends. One possibility is that thisbinding is sufficient to directly hinder the binding of polymerases to theopposite broken DNA. Alternatively, additional factors (perhaps thoseinvolved in DNA repair) may be required to suppress simultaneous bi-directional transcription initiation at DSBs. Further research is neededto elucidate the role, if any, of break-induced RNAs in the DDR, a notionthat was previously suggested (7, 27). Regardless of the function of thesetranscripts, our data are consistent with a model that places bidirectionaltranscription initiation as awidespread outcome ofDSBswithin the bodyof active genes, disclosing a determinant role for chromatin in the func-tional interactions between DNA damage and transcription.


MATERIALS AND METHODSPlasmids and genetic constructsThe plasmid pcDNA5-FRT-TO-IgM-1.7k-PY-CFPPTS-24MS2SL–I–SceI–24PP7SL was constructed with the restriction site for I–SceIinsertedwithin the exon II of amouse immunoglobulinm (IgM) reportergene, referred to as EX2 reporter. The 24 PP7 stem-loop sequencewas excised from pCR4-24xPP7SL [Addgene plasmid no. 31864 (28)]by Bam HI and Bgl II digest and inserted into the Bam HI site ofpCMV5-24xMS2SL, generating pCMV5-24xMS2SL-24xPP7SL. To in-sert the I–SceI restriction site in pCMV5-24xMS2SL-24xPP7SL, twooligo sequences encoding the I–SceI and two BamHI sites were hybrid-ized (sequences are shown in table S1) and amplified by polymerasechain reaction (PCR), purified (NZYGelpure kit, NZYTech), andBam HI digested and ligated into the same site to generate pCMV5-24xMS2SL–I–SceI–24xPP7SL. The Ecl136II– and AleI–generated24xMS2SL-I–SceI–24xPP7SL fragment was cloned into the bluntedXho I site of the pcDNA5-FRT-TO-IgM-1.7k-PY-CFPPTS reporter(10) using the PaperClip protocol (29). The plasmid pcDNA5-FRT-TO-I–SceI–ExI-24bcMS2-IgM-1.7K.PY-CFPPTS was designed withthe I–SceI site in the promoter proximal region of the IgM reportergene, referred to as the PROP reporter. An array of two tet operator(tetO) sequences followed by an I–SceI site, 24 MS2 stem loops fromtwo nonidentical stem-loop sequences [as described for Addgene plas-mid no. 31865 (30)], including five additional nonidentical spacer se-quences, to further decrease redundancywas de novo designed (synthesisby GeneArt, Invitrogen). The 2tetO-(I–SceI)-24×MS2SL fragmentwas cloned by Ecl136II and Hind II digest into the same sites inpcDNA5-FRT-TO-IgM-1.7k-PY-CFPPTS. The third reporter gene re-ferred to as EX2-AS was designed on the basis of the human ubiquitinB gene (HGNC:12463; ENST00000302182.7) with an I–SceI site in ex-on II followed by an open reading frame of two head-to-tail ubiquitinunits (synthesis byGeneArt, Invitrogen). The 24MS2 stem-loop sequencewith five nonidentical spacers [lacking the 2tetO-(I–SceI) part] was li-gated in antisense direction into exon I of the reporter gene and the24 PP7 stem-loop sequence in sense orientation into the exon II 3′ un-translated region. The complete reporter unit was ligated into thepcDNA5-FRT-TO (Invitrogen, Thermo Fisher Scientific) backbone.The plasmids encoding tandem dimers of MS2 and PP7 coat proteins(named MCP and PCP thereafter, respectively) fused to fluorescentproteins were constructed the following way: First, pEGFP-C1 wasVsp I and Bsp 1407I digested, blunted, and religated to remove theCMV promoter and enhanced GFP (EGFP) sequences. The Eco RI–Not I fragment (ubiquitin C promoter) from phage-ubc-nls-ha-tdPCP-gfp [Addgene plasmid no. 40650 (31)] was ligated into the Eco RI–Bsp120I sites generating a pUBC vector. Upstream restriction sites were re-moved by religating blunted EcoRI+BglII sites and EGFP includingmulticloning site were reinserted by ligating a Bsh TI–Xba I EGFP frag-ment from pEGFP-C1 into Cfr9I–Xba I sites in pUBC to create pUBC-GFP-C1. The pUBC-GFP-nls-tdMCP-GFP plasmid (referred to asMS2-GFP) was generated by ligating a Bsp120I+BglII fragment fromphage-ubc-nls-ha-tdMCP-gfp [Addgene plasmid no. 40649 (31)] intoBsp120I+BamHI sites of pUBC-GFP-C1. pUBC-GFP-nls-tdPCP-GFPplasmid (referred to as PP7-GFP)was generated by ligating a Bsp120I+SmaIfragment from phage-ubc-nls-ha-tdPCP-gfp [Addgene plasmid no.40650 (31)] into Bsp120I+KspAI sites of pUBC-GFP-C1. The pUBC-TagRFPt-nls-tdMCP-TagRFPt (referred to as MS2-RFP) was cloned byPCR amplification of the TagRFPt sequence from phage-UBC-nls-ha-2XMCP-TagRFPt [Addgene plasmid no. 64541 (32)] using primerswith AleI and BglII sites (sequences are shown in table S1) to ligate

8 of 12





Dow

nloaded from

it into the same sites of pUBC-GFP-C1 to generate pUBC-TagRFPt.The Bsp 120I+Bam HI fragment containing nls-tdMCP-TagRFPtfrom Addgene plasmid no. 64541 was ligated into the same sites inpUBC-TagRFPt. The pI–SceI–GR-iRFP (near-infrared fluorescentprotein) construct was made by removing RFP from pI–SceI–GR-RFP[Addgene plasmid no. 17654 (33)] by religating Ecl136II+blunted AarIsites, creating an in-frame deletion. Next, the BamHI+BglII fragment frompIRES-H2BiRFP was ligated into the same sites of the I–SceI–GRcontaining plasmid to generate pI–SceI–GR-iRFP.

Stable cell linesThe Flp-In T-REx 293 cell line (Thermo Fischer Scientific) was usedto allow for single genomic integration of the reporter genes by Flprecombinase–mediated integration. Cells were grown as monolayersin high-glucose (4.5 g/liter)Dulbecco’smodifiedEaglemedium (DMEM)supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)L-glutamine (Thermo Fisher Scientific). To create the PROP, EX2,and EX2-AS reporter cells, the pOG44 Flp recombinase expressionvector was cotransfected with the respective reporter construct.Transfected cells were maintained under selection with hygromycinB (200 mg/ml; Roche) and blasticidin (15 mg/ml; InvivoGen) at 37°Cin a humidified atmosphere with 5% CO2.

Live cell experiments and transfectionsWhere indicated, the reporter cell lines were seeded in DMEM withHepes, without phenol red (Gibco) supplemented with 10% (v/v)charcoal-stripped fetal bovine serum (Sigma-Aldrich). For the livecell imaging experiments, cells were plated on 35-mm petri dishes with10-mm glass-bottom microwell (coverglass thickness no. 1.5) (MatTekCorporation). Cells were transiently transfected with one or moreplasmids simultaneously 16 to 24 hours before experiments usingLipofectamine 3000 reagent (Thermo Fisher Scientific), according tothe manufacturer’s protocol. Transcription of the reporter geneswas induced with doxycycline (0.5 mg/ml; D9891, Sigma-Aldrich),where indicated, and nuclear translocation of pI–SceI–GR-iRFP orpI–SceI–GR-RFP was induced by adding TA (T6501, Sigma-Aldrich),prepared in dimethyl sulfoxide (DMSO), and, at the time of treat-ment, diluted in the abovementioned culture medium (final concentra-tion, 10−7 M).

In vitro I–SceI cleavage efficiency assaysTo determine the in vitro cleavage efficiency by the restriction enzymeI–SceI, we first linearized each reporter gene plasmid by a restrictionenzymedigest, followed by enzyme inactivation, and extracted theDNAusing a column purification protocol (NZYTech): The EX2 reporterconstruct was Xho I digested, and the PROP and EX2-AS reporterconstructs were each Hind III digested (all enzymes are from ThermoFisher Scientific). Incubation of the predigested EX2 plasmid withthe I–SceI restriction enzyme (Thermo Fisher Scientific) generated1.5– and 1.2–kilo-bp (kbp) fragments. Aliquots were removed at dif-ferent time points (2.5, 5, 10, and 15 min after incubation with I–SceI).Predigested PROP and EX2-AS plasmids incubated for 15 min withI–SceI generated one additional fragment of 1.5 and 2.0 kbp, respec-tively, and all digests were analyzed on 1% agarose gels, together withnon–I–SceI–digested controls. DNA was visualized by GelRed fluores-cence (Biotium), and agarose gel images were acquired using the Bio-Rad gel imager. The bands were quantified using ImageJ software. Thein vitro I–SceI cleavage efficiency was calculated by measuring thebackground-corrected integrated band intensity of the uncleaved


2.7 kbp and the total of the cleaved 1.5- + 1.2-kbp fragments at theindicated times of I–SceI incubation.

Live cell spinning disk confocal imaging and image analysisLive cell imaging was performed on a 3i Marianas SDC spinning diskconfocal imaging system (Intelligent Imaging Innovations Inc.) using asimilarmicroscopy setup previously described (34). The system is basedon anAxioObserver Z1 invertedmicroscope (Carl ZeissMicroImagingInc., Germany) equipped with a Yokogawa CSU-X1 spinning disk con-focal head (Yokogawa Electric, Tokyo, Japan) and 100-mW solid-statelasers (Coherent Inc., Santa Clara, CA) coupled to an acousto-optictunable filter. The axial position of the sample was controlled with apiezo-driven stage (Applied Scientific Instrumentation, Eugene, OR).Each MatTek dish was placed in an incubation chamber (PeCon P-Set2000, PeConGmbH, Erbach, Germany) mounted on themicroscopestage and connected to CO2 (CO2 module S, PeCon) and humidity(Heating Device Humidity 2000, PeCon) controllers. The wholemicro-scope body excluding lasers, camera, and spinning disk head wasmain-tained inside a large plexiglass environmental chamber (PeCon, Erbach,Germany). The temperature in both the microscope and top stage in-cubation chambers was controlled by a common unit and set to 37°C.The environment inside the top stage incubation chamber was furtherset to 5% CO2 and 100% humidity. Samples were illuminated with l =488 nm for GFP, l = 561 nm for TagRFPt, and l = 640 nm for iRFP.Images were acquired using a 100× (Plan Apo, 1.4 numerical aperture)oil immersion objectives (Carl ZeissMicroImaging Inc.) under the con-trol of SlideBook 6.0 software (Intelligent Imaging Innovations, Denver,CO). Three-dimensional (3D) time-lapse image stacks of 12 to 16 opticalslices separated by 0.4 mm were collected every 30 s for 50 to 60 min,with exposure acquisition times between 40 and 50ms. Digital images(16-bit)were acquiredusingaback thinnedair-cooledelectron-multiplyingcharge-coupled device camera (Evolve 512, Photometrics, Tucson, AZ).3D time-lapse sequences were analyzed with the spot tracking andquantification software tool, STaQTool, as described previously (34, 35).Briefly, this tool was used to track single transcription sites in the cellnucleus over time and calculate the total fluorescence intensity (TFI) foreach diffraction-limited spot in a time-lapse sequence by performing a2D Gaussian fit on the volume of interest at the Z plane correspondingto the highest intensity value (35). ImageJ was used to quantify labelingintensities of a selected ROI defined around the transcription site to de-termine MDC1 accumulation. Briefly, time-lapse frames in both green(GFP) and red (TagRFPt) were first corrected for fluorescence loss dueto photobleaching, as described previously (36). A circular ROI wasused to define the transcription site in the RFP channel. The sameROI was then duplicated in the GFP channel and used to determinethe average intensity over time of MDC1 in the transcription site forthe whole time-lapse sequence.

Microscopy calibration for single transcript detectionCalibration experiments to relate TFI measurements to single moleculeand single transcript numbers were carried out as follows: HEK293 cellswere transfected with pEGFP-C1 (Clontech) and harvested on ice thenext day. Cytoplasmic extract was prepared by swelling and lysing cellsin RSB (resuspension buffer) (10 mM tris, 10 mMNaCl, and 3mMMgCl2). Cytoplasmic extract was then diluted in 1× phosphate-bufferedsaline (PBS) at 1:50 to 1:1000, and 1 to 2 ml were placed betweentwo 30-mm no. 1.5 coverslips (Thermo Fisher Scientific) and placedin a PeCon POCmini-2 cell chamber as described (34). The chamberwas placed in the same stage incubator as described above, and GFP

9 of 12





Dow

nloaded from

molecules were imaged with the same laser intensity as in the live cellexperiments butwith exposure times of 1000ms over 100 time points inone plane. Image series were analyzed using the StaQTool to detect flu-orescent objects and measure TFI values. Single GFP molecules wereidentified by detecting events irreversible and rapid loss of fluorescencebleaching, where the corresponding difference of the TFI value wasfitted and recorded as TFI of a single GFP molecule (fig. S4A). Live cellexperiments to determine the labeling rate of single reporter gene tran-scripts were performedusingPROPandEX2 reporter cell lines transient-ly transfected with MS2-GFP or PP7-GFP, respectively. One day aftertransfection, the reporter gene transcription was induced with doxycycline(0.5 mg/ml) for 1 hour, and single imaging plane time series of 100 timepointswith 500-ms intervals were recorded. Particleswith highmobilityin the cell nucleus represent single-labeled mRNA transcripts in con-trast to stationary larger objects representing sites of reporter genetranscription with multiple-labeled RNAs present. Analysis of thebrightness of single-labeled reporter gene mRNAs was performedusing the STaQTool by determining the TFI of single-labeled mRNAmolecules in the image frame (fig. S4B). The mean TFI value of singleGFP molecules was related to the mean TFI of MS2-GFP– or PP7-GFP–labeled transcripts to determine the labeling ratio (fig. S4C). Ad-ditional live and fixed cell calibration measurements to detect singlemRNA transcripts labeled byMS2-GFP,MS2-RFP, and PP7-GFPwereperformed accordingly to determine the single transcript labelingmeanTFI values for the respective labeling protein and imaging settingsused in the live cell I–SceI cutting assays (fig. S5). Particles with highmobility in the cell nucleus represent single-labeled mRNA transcriptsin contrast to stationary larger objects representing sites of reporter genetranscription with multiple-labeled RNAs present (fig. S5, A to D). Toimprove the TFI measurements of double-labeled transcripts from theEX2 reporter gene, cells were double transfected (MS2-RFP and PP7-GFP) and, after transcription induction for 1 hour, fixed for 10 min in3.7% formaldehyde, washed in PBS, and embedded in VECTASHIELDmounting medium (Vector Laboratories). Image stacks of 30 to 40optical slices at 0.27-mm intervals were taken using the same imagingconditions as in live cell experiments. Analysis was performed usingthe STaQTool by determining the TFI of single-labeled mRNA mole-cules in the image frame (live cells) or plane (fixed cells) of highest in-tensity (fig. S5, E and F) in cells, where the site of reporter genetranscription was identified as a much larger and brighter spot withinthe nucleus and excluded from analysis. Our results show that around13 (MS2) to 15 (PP7) of 24 RNA stem loops were occupied, on average,and confirm a less than 100% labeling ratio detected before.

Chromatin immunoprecipitationChIP was performed as described (14, 37). Briefly, EX2 and PROP cellswere transfected with I–SceI–GR-RFP and, 24 hours later, treated withTA or DMSO (nontreated) as mentioned above. Then, cells were cross-linked in 1% (v/v) formaldehyde for 10min and subsequently quenchedwith 250 mM glycine for 5 min. Precleared chromatin was incubatedwith 3 mg of a specific histone H3 antibody (ab1791, Abcam), H3K36me3(ab9050, Abcam), or H3K9Ac (ab4441, Abcam) overnight at 4°C. One-tenth of the sample was taken aside as input control. Immunoprecipi-tated (IP) DNAwere subsequently purified and quantified. The relativeoccupancy of the IP protein at the specified DNA sites was estimated byreal-time quantitative PCR (RT-qPCR) as follows: 2^(Ct input − Ct IP),whereCt input andCt IP aremean threshold cycles of RT-qPCRdone induplicate on DNA samples from input and specific immunoprecipita-tions, respectively. Data were normalized against the ChIP values ob-


tained in control cells (i.e., without TA). Primer sequences are shownin table S1.

MNase assayThis assay was performed as described (14), with some modifications.Briefly, EX2 cells were prepared as described for the ChIP protocol and,after TA incubation, collected by trypsinization. Cells were resuspendedin ice-cold RSGB40 buffer [10 mM tris (pH 7.4), 10 mM NaCl, 3 mMMgCl2, 10%glycerol, and 0.5%NP-40] and centrifuged (3min, 7000 rpmat 4°C) to isolate nuclei. Nuclei were washed with digestion buffer[10 mM tris (pH 7.4), 15 mM NaCl, 60 mM KCl, and 1 mM CaCl2],centrifuged (3 min, 7000 rpm at 4°C), and resuspended in digestionbuffer. One-half was digested with 10 U of MNase (Fermentas) for30min at 37°C before incubation with stop buffer [100mMEDTA andproteinase K (20 mg/ml)] for 30min at 55°C, and another half was trea-ted with buffer alone (nondigested). Mononucleosome-sized DNA ob-tained after 30-min digestion and nondigested DNA were used afterstandard phenol-chloroform purification as templates for RT-qPCR.The amount of MNase-resistant DNA at each gene specific regionwas estimated as follows: 2^(Ct nondigested – Ct MNase digested),where Ct t0 and Ct t30 are the mean threshold cycles of RT-qPCR donein duplicate on DNA samples from nondigested (t0) and 30-minMNase-digested (t30) samples, respectively. Data were normalizedagainst the MNase values were obtained in control cells (i.e., withoutTA). Primer sequences are shown in table S1.

RNA interferenceCells were seeded into p35 dishes and simultaneously transfected with5 ml of 10 mM duplex siRNAs (sequences given in table S1) (TriFECTaKit DsiRNA Duplex, Integrated DNA Technologies) using Opti-MEM(Invitrogen) and Lipofectamine RNAiMAX (Invitrogen), according tothe manufacturer’s instructions. The following day, cells were split andseeded into two glass-bottom microscopy dishes in the medium indi-cated above for live cell imaging experiments and transfected again withsiRNAusing the same protocol. After 8 hours, around 30% of the growthmedium was exchanged, and the cells were transfected with the plas-mids encoding fluorescent proteins as indicated above.

Western blotWhole-cell protein extracts were prepared by cell lysis with SDS–polyacrylamide gel electrophoresis (PAGE) buffer [80 mM tris-HCl(pH 6.8), 16% glycerol, 4.5% SDS, 450 mM dithiothreitol, and 0.01%bromophenol blue], with benzonase (200 U/ml; Sigma-Aldrich) and50 mMMgCl2 and boiling for 5min. Equal amounts of protein extractswere resolved by SDS-PAGE and transferred to a nitrocellulose mem-brane. Immunoblotting was performed with the following antibodies:rabbit anti-DNA ligase IV (ab193353, Abcam), mouse anti–a-tubulin(T5168, Sigma-Aldrich), rabbit anti–total histone H3 (ab1791, Abcam)and using the secondary antibodies goat anti-mouse horseradish perox-idase (HRP) (170-6516, Bio-Rrad) and goat anti-rabbitHRP (170-6515,Bio-Rad).

Statistical analysisWhere indicated, data were subjected to a two-tailed Student’s t test toresolve statistical significance. The measured TFI values of GFP mole-cules and single-labeled mRNA transcripts were plotted into histo-grams and fitted with a Gaussian function according to the formula

y ¼ y0 þ Aw

ffiffiffiffiffi

p=2p e�2ðx�xcÞ2

w2 to determine themean value xc of the intensity

10 of 12





Dow

nloaded from

distribution in OriginPro 8. The mean TFI value for single GFP mole-cules was related to themean single transcript intensities via conversionof the exposure time at identical imaging settings. The comparison ofDNA ligase IV knockdown and control live cell experiment datasetswas performed using Fisher’s exact test for 2 × 2 contingency tablesand a c2 test for contingency tables, both at a level of significance ofP = 0.05.

Bioinformatics analysesEndogenous DSBs on NHEK cells were obtained from a previousstudy (18), considering only the high-confidence 84,946 DSBs com-mon to DSBCapture replicates. Transcription was assessed usingpolyA-depleted and strand-specific RNA sequencing (RNA-seq)data from nuclear fractions of NHEK cells (GSM2072453). DNAaccessibility profiles (DNase-seq) for NHEK cells were previouslyidentified by the National Institutes of Health Roadmap EpigenomicsMapping Consortium. DSB and DNA accessibility coordinates wereconverted from hg19 into hg38 human genome versions using liftOver(38). Gene annotations were obtained from GENCODE (v26 version),merged into a single transcript model per gene and removedoverlapping genes using BEDtools (39). Only DSBs located in the genebody region [500 bp downstream TSS (transcription start-site) to TTS(transcription termination-site)] of transcriptionally active genes wereconsidered in the downstream analyses. Transcriptionally active geneswere defined as those with expression levels [TPMs (transcripts permillion)] from Kallisto (40) higher than the 50th percentile (median).DSBs with antisense transcription were determined with a cutoff of0.01 RPKMs (reads per kilobase per million mapped reads) in the500-bp flanking regions, according to the strand-specific information.For themetaprofiles, DSBs were aligned by themedian region, and theread density for the flanking 1 kbwas averaged in a 20-bpwindow. Allprofiles and heatmaps were plotted on normalized RPKMs considering20-bpwindows. A set of in-house scripts for data processing and graph-ical visualization were written in bash and in the R environmentallanguage (www.R-project.org). SAMtools (41) and BEDtools were usedfor alignment manipulation, filtering steps, file format conversion, andcomparison of genomic features. Fold enrichment over random andstatistical significance of the overlap between transcription andDSBswas assessed by permutation analysis. Briefly, randomDSBs datasetswere generated 1000 times from transcriptionally active genes usingthe shuffle BEDtools function (maintaining the number and lengthof the original datasets). The P value was determined as the frequencyof overlapping regions between the random datasets as extreme as theobserved data.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau1249/DC1Fig. S1. Schematics of the reporter genes.Fig. S2. Detection of single RNAs and measurement of transcription rates.Fig. S3. I–SceI cleaves the reporter genes driving MDC1 recruitment to damaged loci.Fig. S4. Single-molecule calibration measurements.Fig. S5. Single transcript calibration measurements.Fig. S6. Break-induced nucleosome depletion.Table S1. Sequence of primers used in this study.

REFERENCES AND NOTES1. Y. W. Fong, C. Cattoglio, R. Tjian, The intertwined roles of transcription and repair

proteins. Mol. Cell 52, 291–302 (2013).2. H. Gaillard, A. Aguilera, Transcription as a threat to genome integrity. Annu. Rev. Biochem.

85, 291–317 (2016).


3. B.-G. Ju, V. V. Lunyak, V. Perissi, I. Garcia-Bassets, D. W. Rose, C. K. Glass, M. G. Rosenfeld, Atopoisomerase IIb-mediated dsDNA break required for regulated transcription. Science312, 1798–1802 (2006).

4. R. Madabhushi, F. Gao, A. R. Pfenning, L. Pan, S. Yamakawa, J. Seo, R. Rueda, T. Phan,H. Yamakawa, P.-C. Pao, R. T. Stott, E. Gjoneska, A. Nott, S. Cho, M. Kellis, L.-H. Tsai,Activity-induced DNA breaks govern the expression of neuronal early-response genes.Cell 161, 1592–1605 (2015).

5. H. Bunch, B. P. Lawney, Y.-F. Lin, A. Asaithamby, A. Murshid, Y. E. Wang, B. P. C. Chen,S. K. Calderwood, Transcriptional elongation requires DNA break-induced signalling. Nat.Commun. 6, 10191 (2015).

6. J. Puc, A. K. Aggarwal, M. G. Rosenfeld, Physiological functions of programmed DNAbreaks in signal-induced transcription. Nat. Rev. Mol. Cell Biol. 18, 471–476 (2017).

7. F. Michelini, S. Pitchiaya, V. Vitelli, S. Sharma, U. Gioia, F. Pessina, M. Cabrini, Y. Wang,I. Capozzo, F. Iannelli, V. Matti, S. Francia, G. V. Shivashankar, N. G. Walter, F. d’Adda di Fagagna,Damage-induced lncRNAs control the DNA damage response through interaction withDDRNAs at individual double-strand breaks. Nat. Cell Biol. 19, 1400–1411 (2017).

8. M. Vera, J. Biswas, A. Senecal, R. H. Singer, H. Y. Park, Single-cell and single-moleculeanalysis of gene expression regulation. Annu. Rev. Genet. 50, 267–291 (2016).

9. X. Darzacq, Y. Shav-Tal, V. de Turris, Y. Brody, S. M. Shenoy, R. D. Phair, R. H. Singer, In vivodynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806 (2007).

10. R. M. Martin, J. Rino, C. Carvalho, T. Kirchhausen, M. Carmo-Fonseca, Live-cell visualizationof pre-mRNA splicing with single-molecule sensitivity. Cell Rep. 4, 1144–1155 (2013).

11. G. S. Stewart, B. Wang, C. R. Bignell, A. M. R. Taylor, S. J. Elledge, MDC1 is a mediator of themammalian DNA damage checkpoint. Nature 421, 961–966 (2003).

12. Z. Lou, K. Minter-Dykhouse, X. Wu, J. Chen, MDC1 is coupled to activated CHK2 inmammalian DNA damage response pathways. Nature 421, 957–961 (2003).

13. T. E. Wilson, U. Grawunder, M. R. Lieber, Yeast DNA ligase IV mediates non-homologousDNA end joining. Nature 388, 495–498 (1997).

14. S. Carvalho, A. C. Raposo, F. B. Martins, A. R. Grosso, S. C. Sridhara, J. Rino,M. Carmo-Fonseca, S. F. de Almeida, Histone methyltransferase SETD2 coordinates FACTrecruitment with nucleosome dynamics during transcription. Nucleic Acids Res. 41,2881–2893 (2013).

15. C. D. Kaplan, L. Laprade, F. Winston, Transcription elongation factors repress transcriptioninitiation from cryptic sites. Science 301, 1096–1099 (2003).

16. S. Marquardt, R. Escalante-Chong, N. Pho, J. Wang, L. S. Churchman, M. Springer,S. Buratowski, A chromatin-based mechanism for limiting divergent noncodingtranscription. Cell 157, 1712–1723 (2014).

17. I. Whitehouse, O. J. Rando, J. Delrow, T. Tsukiyama, Chromatin remodelling at promoterssuppresses antisense transcription. Nature 450, 1031–1035 (2007).

18. S. V. Lensing, G. Marsico, R. Hänsel-Hertsch, E. Y. Lam, D. Tannahill, S. Balasubramanian,DSBCapture: In situ capture and sequencing of DNA breaks. Nat. Methods 13, 855–857(2016).

19. N. M. Shanbhag, I. U. Rafalska-Metcalf, C. Balane-Bolivar, S. M. Janicki, R. A. Greenberg,ATM-dependent chromatin changes silence transcription in cis to DNA double-strandbreaks. Cell 141, 970–981 (2010).

20. F. Iannelli, A. Galbiati, I. Capozzo, Q. Nguyen, B. Magnuson, F. Michelini, G. D’Alessandro,M. Cabrini, M. Roncador, S. Francia, N. Crosetto, M. Ljungman, P. Carninci, F. d’Adda di Fagagna,A damaged genome’s transcriptional landscape through multilayered expression profilingaround in situ-mapped DNA double-strand breaks. Nat. Commun. 8, 15656 (2017).

21. A. Ui, Y. Nagaura, A. Yasui, Transcriptional elongation factor ENL phosphorylated by ATMrecruits polycomb and switches off transcription for DSB repair. Mol. Cell 58, 468–482(2015).

22. A. Kakarougkas, A. Ismail, A. L. Chambers, E. Riballo, A. D. Herbert, J. Künzel, M. Löbrich,P. A. Jeggo, J. A. Downs, Requirement for PBAF in transcriptional repression and repair atDNA breaks in actively transcribed regions of chromatin. Mol. Cell 55, 723–732 (2014).

23. T. Pankotai, C. Bonhomme, D. Chen, E. Soutoglou, DNAPKcs-dependent arrest of RNApolymerase II transcription in the presence of DNA breaks. Nat. Struct. Mol. Biol. 19,276–282 (2012).

24. S. W. Awwad, E. R. Abu-Zhayia, N. Guttmann-Raviv, N. Ayoub, NELF-E is recruited to DNAdouble-strand break sites to promote transcriptional repression and repair. EMBO Rep.18, 745–764 (2017).

25. M. Kruhlak, E. E. Crouch, M. Orlov, C. Montaño, S. A. Gorski, A. Nussenzweig, T. Misteli,R. D. Phair, R. Casellas, The ATM repair pathway inhibits RNA polymerase I transcription inresponse to chromosome breaks. Nature 447, 730–734 (2007).

26. B. D. Price, A. D. D’Andrea, Chromatin remodeling at DNA double-strand breaks. Cell 152,1344–1354 (2013).

27. I. Schmidts, R. Böttcher, M. Mirkovic-Hösle, K. Förstemann, Homology directed repair isunaffected by the absence of siRNAs in Drosophila melanogaster. Nucleic Acids Res. 44,8261–8271 (2016).

28. D. R. Larson, D. Zenklusen, B. Wu, J. A. Chao, R. H. Singer, Real-time observation oftranscription initiation and elongation on an endogenous yeast gene. Science 332,475–478 (2011).

11 of 12

http://www.R-project.org

http://advances.sciencemag.org/cgi/content/full/5/1/eaau1249/DC1

http://advances.sciencemag.org/cgi/content/full/5/1/eaau1249/DC1



http://advaD

ownloaded from

29. M. Trubitsyna, G. Michlewski, Y. Cai, A. Elfick, C. E. French, PaperClip: Rapid multi-part DNAassembly from existing libraries. Nucleic Acids Res. 42, e154 (2014).

30. E. Bertrand, P. Chartrand, M. Schaefer, S. M. Shenoy, R. H. Singer, R. M. Long, Localizationof ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

31. B. Wu, J. A. Chao, R. H. Singer, Fluorescence fluctuation spectroscopy enables quantitativeimaging of single mRNAs in living cells. Biophys. J. 102, 2936–2944 (2012).

32. J. M. Halstead, T. Lionnet, J. H. Wilbertz, F. Wippich, A. Ephrussi, R. H. Singer, J. A. Chao,Translation. An RNA biosensor for imaging the first round of translation from single cellsto living animals. Science 347, 1367–1671 (2015).

33. E. Soutoglou, J. F. Dorn, K. Sengupta, M. Jasin, A. Nussenzweig, T. Ried, G. Danuser,T. Misteli, Positional stability of single double-strand breaks in mammalian cells. Nat. CellBiol. 9, 675–682 (2007).

34. R. M. Martin, J. Rino, A. C. de Jesus, M. Carmo-Fonseca, Single-molecule live-cellvisualization of pre-mRNA splicing. Methods Mol. Biol. 1358, 335–350 (2016).

35. J. Rino, A. C. de Jesus, M. Carmo-Fonseca, STaQTool: Spot tracking and quantification toolfor monitoring splicing of single pre-mRNA molecules in living cells. Methods 98, 143–149(2016).

36. R. D. Phair, T. Misteli, High mobility of proteins in the mammalian cell nucleus. Nature404, 604–609 (2000).

37. S. C. Sridhara, S. Carvalho, A. R. Grosso, L. M. Gallego-Paez, M. Carmo-Fonseca,S. F. de Almeida, Transcription dynamics prevent RNA-mediated genomic instabilitythrough SRPK2-dependent DDX23 phosphorylation. Cell Rep. 18, 334–343 (2017).

38. A. S. Hinrichs, D. Karolchik, R. Baertsch, G. P. Barber, G. Bejerano, H. Clawson, M. Diekhans,T. S. Furey, R. A. Harte, F. Hsu, J. Hillman-Jackson, R. M. Kuhn, J. S. Pedersen, A. Pohl,B. J. Raney, K. R. Rosenbloom, A. Siepel, K. E. Smith, C. W. Sugnet, A. Sultan-Qurraie,D. J. Thomas, H. Trumbower, R. J. Weber, M. Weirauch, A. S. Zweig, D. Haussler, W. J. Kent,The UCSC Genome Browser Database: Update 2006. Nucleic Acids Res. 34, D590–D598(2006).

39. A. R. Quinlan, I. M. Hall, BEDTools: A flexible suite of utilities for comparing genomicfeatures. Bioinformatics 26, 841–842 (2010).

40. N. L. Bray, H. Pimentel, P. Melsted, L. Pachter, Near-optimal probabilistic RNA-seqquantification. Nat. Biotechnol. 34, 525–527 (2016).


41. H. Li, B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. Marth, G. Abecasis,R. Durbin; 1000 Genome Project Data Processing Subgroup, The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

Acknowledgments: We thank R.H. Singer, J.A. Chao, T. Misteli, M. Carmo-Fonseca, E. Gomes,J. Lukas, and C. M Azzalin for comments and gifts of plasmids and reagents. We are alsoindebted to J. Rino, A. Temudo, and A. Nascimento from the iMM Bioimaging Facility andS. Carvalho for excellent technical support. Funding: This work was funded by PTDC/BIM-ONC/0016/2014, PTDC/MED-OUT/32271/2017, and PTDC/BIA-MOL/30438/2017 from Fundação paraa Ciência e a Tecnologia (FCT), Portugal and by LISBOA-01-0145-FEDER-007391, projectcofunded by FEDER through POR Lisboa, Portugal 2020–Programa Operacional Regional deLisboa, and FCT. A.R.G. is the recipient of a FCT Investigator grant (IF/00510/2014). A.C.V. andJ.C.S. are recipients of FCT PhD fellowships SFRH/BD/52232/2013 and PD/BD/128292/2017.Author contributions: A.C.V. performed most cell culture, microscopy, and biochemicalexperiments and acquired, analyzed, and interpreted data. S.C.S. and J.C.S. performedbiochemical assays and analyzed data. A.I.A. performed cloning experiments. A.R.G. analyzedand interpreted RNA-seq, DSBCapture, and DNase-seq data. R.M.M. generated the reportergenes, performed microscopy experiments, and acquired, analyzed, and interpreted data.S.F.d.A. conceived the study, analyzed and interpreted data, and wrote the manuscript with theassistance of all authors. Competing interests: The authors declare that they have no competinginterests. Data and materials availability: All data needed to evaluate the conclusions in thepaper are present in the paper and/or the Supplementary Materials. Additional data related to thispaper may be requested from the authors.

Submitted 9 May 2018Accepted 29 November 2018Published 9 January 201910.1126/sciadv.aau1249

Citation: A. C. Vítor, S. C. Sridhara, J. C. Sabino, A. I. Afonso, A. R. Grosso, R.M.Martin, S. F. de Almeida,Single-molecule imaging of transcription at damaged chromatin. Sci. Adv. 5, eaau1249 (2019).

nc

12 of 12


es.sciencemag.org/


Single-molecule imaging of transcription at damaged chromatin

AlmeidaAlexandra C. Vítor, Sreerama C. Sridhara, João C. Sabino, Ana I. Afonso, Ana R. Grosso, Robert M. Martin and Sérgio F. de

DOI: 10.1126/sciadv.aau1249 (1), eaau1249.5Sci Adv

ARTICLE TOOLS http://advances.sciencemag.org/content/5/1/eaau1249

MATERIALSSUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2019/01/07/5.1.eaau1249.DC1

REFERENCES

http://advances.sciencemag.org/content/5/1/eaau1249#BIBLThis article cites 41 articles, 5 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.Science AdvancesYork Avenue NW, Washington, DC 20005. The title (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 NewScience Advances

License 4.0 (CC BY-NC).Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of



Dow

nloaded from

http://advances.sciencemag.org/content/5/1/eaau1249

http://advances.sciencemag.org/content/suppl/2019/01/07/5.1.eaau1249.DC1

http://advances.sciencemag.org/content/5/1/eaau1249#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


Documents

Copyright © 2019 Single-molecule imaging of transcription ... · (PROP) region downstream the transcription start site and a region within an internal exon (EX2). Induction of a