Histone modification in living cells

1Department of Plant Biotechnology03/11/2016


Contents

Histones are a group of basic protein that associate with DNA and help the DNA to condense it into chromatin.

Histones contain a large proportion of the positively charged (basic) amino acids, lyseine and arginine in their structure.

DNA is negatively charged due to the phosphate groups on its backbone.

These result of these opposite charges is strong attraction and therefore high binding affinity between histones and DNA structure called nucleosome.

DNA wraps around histones, they also play a role in gene regulation.

3Department of Plant Biotechnology

Luger et al., Nature 1997

Principles of Biochemistry, Lehninger 4th Edition.

03/11/2016

Introduction

Histones

The basic unit of chromatin is the nucleosome core particle, which contains 147 bp of DNA wrapped nearly twice around an octamer of the core histones.

Each nucleosome is separated by 10–60 bp of ‘linker’ DNA, and the resulting nucleosomal array constitutes a chromatin fiber of ~10 nm in diameter.


Genome Organization

Mol. Bio of Gene, Watson et al., 7th edition

Luger et al., Nature 1997

Histone proteins are of two types:

Core Histones - H2A, H2B, H3, and H4 Linker Histones - H1


The eight histones in the core are arranged into a (H3)2(H4)2 tetramer and a pair of H2A–H2B dimers.

The tetramer and dimers come together to form a left-handed superhelical ramp around which the DNA wraps.

Hydrogen bonds between the DNA backbone and the amide group on the main chain of histone proteins

03/11/2016

Types of Histones

Mol. Bio of Gene, Watson et al., 7th edition

Department of Plant Biotechnology 6

Histone H1 is not part of the nucleosome core particle. Instead, it binds to the linker DNA and is referred to as a linker histone.

H1 is half as abundant as the other histones, which is consistent with the finding that only one molecule of H1 can associate with a nucleosome.

Since H2A packages DNA molecules into chromatin, the packaging process will effect gene expression.

Refers to a variety of closely related proteins that vary often by only a few amino acids.

H2A plays a major role in determining the overall structure of chromatin. Inadvertently, H2A has been found to regulate gene expression.

H2B is also involved with the structure of the nucleosomes of the 'beads on a string' structure.

03/11/2016

H1

H2A

H2B

http://www.actrec.gov.in/histome/histone_main.php


H4 is a structural component of the nucleosome, and is subject to covalent modification ,including acetylation and methylation, which may alter expression of genes located on DNA associated with its parent histone octamer.

Featuring a main globular domain and a long N-terminal tail.

Also an important protein in the emerging field of epigenetics, where its sequence variants and variable modification states are thought to play a role in the dynamic and long term regulation of genes.

03/11/2016

H4

H3

http://www.actrec.gov.in/histome/histone_main.php


The core histones each have an amino-terminal extension, called a tail because it lacks a defined structure and is accessible within the intact nucleosome.

These tails are rich in number of lysine (K) and arginine (R) residues.

The C-terminal end is primarily responsible for histone-DNA and histone-histone interactions.

The N-terminal tails stand as targets of post-transational modifications (PTMs), which may modify the structure of chromatin play an essential role in regulating gene expression.

© J.H.Waterborg,1999

© Maacmillan pub.Luger et al., 199703/11/2016

(g/mol)


N-terminal tails of histones are the most accessible regions of these peptide as they protrude from the nucleosome and possess no specific structure.

The major function of PTMs is to either create sites for the recruitment of specific factors or modify existing sites so as to abolish previous interactions.

Chromatin must be first made relaxed to allow access of cellular machineries to chromatin DNA.

The amino-terminal portion of the core histone proteins contains a flexible and highly basic tail region, which is conserved across various species and is subject to various PTM.

Chromatin can be highly packed or loosely packed, and correlated to the gene expression levels.

Post-translational modification (PTM) of histones is a crucial step in epigenetic regulation of a gene.

03/11/2016

N-termini of the core histones

©1999, J.H.Waterborg, UMKC

Zdenko Herceg1 and Rabih Murr1,2

Histone Modification


Modifications in histone proteins affects the structure of chromatin.

Gene regulation

DNA damage and repair

Chromosome condensation

Heterochromatin

-Highly packed form- Low gene expression

Euchromatin

- Loosely packed form- High gene expression

03/11/2016

N-terminal tails of all histones are particularly of interest since they protrude out of the compact structure. These N-terminal tails are often subjected to a variety of post-translational modifications such as,


Methyl Acetyl Phospho Ubiquitin SUMO

Bhaumik, Smith, and Shilatifard, 2007.

It has been proposed that these modifications result in a ‘code’ which can be read by proteins involved in gene expression and other DNA translations

03/11/2016

Types of Histone Modification


Amino acid Residue

Modification Type

Modiying Enzyme

Lysine AcetylationDeacetylation

HAT HDAC

Lysine MethylationDemethylation

HMT HDM

Lysine UbiquitylationDeubiquitylation

Ub ligaseUb protease

Serine/ThreoninePhosphorylationDephosphorylation

KinasePhosphatase

Arginine MethylationDemethylation

PRMTDeiminase/Demethylase

Some Examples of Histone Modification and Modifiers

Others: Sumoylation (Lysine), ADP Ribosylation (Glutamate)

Post-translational Modification of Histone N-terminal Tails

03/11/2016


N-term tails reversible acetylated in Lys, particularly in H3+H4

While the globular core is involved in histone-histone packing and DNA-contact, the N-terminal tails point outwards and is available for interaction.

Acetylation also provides binding sites for a number of proteins with an approximately 100-amino-acid sequence motif called a bromodomain.

++++ +

Luger et al., 1997

03/11/2016

1. Acetylation

P.J. Barnes et al., Eur Respir J 2005


Histone acetylation Histone deacetylation

Enzyme Histone acetyl transferases (HATs)

Histone deacetylases (HDACs)

Group Adds acetyl groups to histone tails

Removes acetyl groups from histone tails

Interacion with DNA

Reduces positive charge and weakens interaction of histones with DNA

Increases interaction of DNA and histones

©2006 Prous Science

03/11/2016

Acetylation Deacetylation

Role in Gene regulation

Allows transcription Repress transcription

Acetyl co.A


Modification Histone Site Possible function

Acetylation H2A K4, K5,K7 Transcriptional activation

H2BK5, K11, K12, K15, K16 , K20 Transcriptional activation

H3 K4, K9, K23, K27 Transcriptional activation

K14Transcriptional activation, DNA repair, Transcription elongation

K18 Transcriptional activation, DNA repair

H4 K5, K12, K16 Transcriptional activation, DNA repair

K8 Transcriptional activation

Sites of histone acetylation with their function-

03/11/2016 P.J. Barnes et al., Eur Respir J 2005

03/11/2016 Department of Plant Biotechnology 16

Role of Acetylation in Gene Expression


It is the introduction of an Methyl functional group to only on Lysine or Arginine of the histone tail.

These reactions are catalyzed by enzymes with "histone methyltransferases (HMTs)”

Histone lysine methyl transferases (HKMTs) Methylate lysine (K) residues

Protein argenin methyl transferases (PRMTs) Methylate arginine (R) residues

A role in both activation and repression

Arginines can be mono- or dimethylated whereas lysines can be mono-, di- or trimethylated .

Arg

Lys

03/11/2016

2. Methylation

Eric J. Richards., et al., 2002


Methylation can result in activation or repression of expression . Activation (H3K4, H3K36, H3K79) Trimethylation of histone H3 at lysine 4 (H3K4) is an universal active mark for transcription.

Repression (H3K9, H3K27, H4K20) Dimethylation of histone H3 at lysine 9 (H3K9) and at 27 (H3K27) are the universal signal for

transcriptional silencing.

Modification Histone Site Possible Function

Methylation H3 K4Permissive euchromatin (di-Me), Active euchromatin (tri-Me)

Transcriptional activation

K9Transcriptional silencing (tri-Me), DNA methylation (tri-Me), Heterochromatin formation

R17 Transcriptional activation

K27 Transcriptional silencing, X inactivation (tri-Me)

K36 Transcriptional elongation

K79 Euchromatin, Transcriptional elongation

H4 R3 Transcriptional activation

K20Transcriptional silencing (mono-Me), Heterochromatin (tri-Me).

K59 Transcriptional silencing

Sites of histone methylation with their function

03/11/2016http://www.actrec.gov.in/histome/histones.php?histone=H3


Model for Role of Methylation in Heterochromatin Formation

Condensation assisted by recruitment of HMT (histone methyltransferase), where HP1 (heterochromatin protein 1) binds to H3K9-Me3 which methylates adjacent H3K9.

Chromatin condensed until a boundary element is reached and turned into heterochromatin

Methylation of histone tails long lasting compared to acetylation.

Can be Inherited by daughter cells: Responsible for X-inactivation in female.

Phillips, T. (2008) Nature Education

03/11/2016


Phosphorylation is the addition of a phosphate group (PO43−) to a molecule.

Phosphorylation is catalyzed by various specific protein kinases, whereas phosphatases mediate removal of the phosphate group.

Histones can also get phosphorylated and the most studied sites of histone phosphorylation are the serine 10 of histone H3 (H3S10) that is deposited by the Aurora-B kinase during mitosis.

Phosphorylation of histones, in particular phosphorylation of H2AX, has a role in DNA damage response and DNA repair.

Rapid phosphorylation of H2AX, at serine 129 (H2AX) by the PI3K kinases at double strand break (DSB) sites, is one of the first and most easily detectable DNA damage signaling post-translational events.

Role of histone phosphorylation in DNA repair

03/11/2016

3. Phosphorylation

Dorine Rossetto, et. al., Epigenetics 7:10 2012



.Phosphorylation H2A S1

Mitosis, Transcriptional repression, Chromatin assembly

T119 MitosisS129 (S. cerevisiae) DNA repairS139 (mammalian H2AX) DNA repair

H2BS14 (vertebrates) Apoptosis

S33 (D. melanogaster) Transcriptional activation

H3 T3 Mitosis

S10Mitosis, meiosis, Transcriptional activation

T11 (mammals) MitosisS28 (mammals) Mitosis

H4 S1 Mitosis

Role of histone phosphorylation in transcription regulation

The phosphorylation of H3S10 (H3S10P) was initially linked to chromosome relaxation and segregation during mitosis and meiosis.

The role of H3S10P in chromatin condensation suggests that it should be involved in transcriptional activation.

Transient derepression by histone H3 phosphorylation. Mitogen-activated protein kinases (MAPK)

Signal molecule

absent

Signal molecule present

Sites of histone phosphorylation with their function

03/11/2016 Anna Sawicka et al., Biochimie 94 (2012)

Ubiquitination (or ubiquitylation) refers to the post-translational modification of the amino group of a lysine residue by the covalent attachment of one (monoubiquitination) or more (polyubiquitination) ubiquitin monomers.

Ubiquitin is a 76 amino acid protein highly conserved in eukaryotes.

Histone ubiquitination alters chromatin structure and allows the access of enzymes involved in transcription

Ubiquitination is carried out in three main steps: activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively.

22Department of Plant BiotechnologyJian Cao et.al.,Frontiers in oncology

03/11/2016

4. Ubiquitination


Ubiquitylation H2AK119 (mammals) Spermatogenesis

H2BK120 (mammals) Meiosis

K123 (S. cerevisiae)

Transcriptional activation

Sites of histone ubiquitination with their function

Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are attached to and detached from other proteins in cell to modify their function.

Sumoylation consists in the addition of a “Small Ubiquitin-related MOdifier protein” (SUMO) of ~100 amino acids.

Histone sumoylation was first reported in 2003, when Shiio et al. found that H4 can be modified by SUMO and they suggested that this modification leads to the repression of transcriptional activity through the recruitment of HDACs and HP1 proteins

The putative sumoylation sites were identified as K6/7 and to a lesser extent K16/17 of H2B, K126 of H2A, and all four lysines in the N-terminal tail of H4.

Histone sumoylation has a role in transcription repression by opposing other active marks such as acetylation, methylation, ubiquitination etc.

23Department of Plant BiotechnologyShiio et. al., PNAS, 2003

03/11/2016

5. Sumoylation

http://www.pnas.org/content/100/23/13118/F1.expansion.html

ADP-ribosylation is the addition of an ADP-ribose moiety onto a protein using NAD+ as a substrate.

Mono(ADP-ribosyl)ation is mediated by ADP ribosyl transferases (ART) and the enzymes responsible for the PARation (Poly-ADP-ribosylation) are the poly(ADP-ribose) polymerases (PARPs).

PARP1 prefers to linker histone H1 while PARP2 prefers core histones.


Zdenko Herceg & Rabih MurrSascha Beneke* www.frontiersin.org, 2012

03/11/2016

6. ADP-Ribosylation

FIGURE 1 | Poly(ADP-ribose) polymerase1 in DNA repair


H1 to nucleosomes increases chromatin compaction, ADP-ribosylation of H1 is suggested to alter the chromatin structure and possibly the chromatin composition.

Messner and Michael , Trends in Cell Biology September 201103/11/2016

1

2

3

ADP-ribosylation cycle of histones in chromatin

Suggested biological consequences of mono- or poly-ADP-ribosylated histones in chromatin.


The histone code is a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends .

This hypothesis predicts that-

•Distinct modifications of the histone tails will induce interaction affinities for chromatin-associated proteins.

•Modifications may be interdependent generate various combinations

•Local concentrations and combinations of differently modified nucleosomes determine qualities of higher order chromatin.

03/11/2016

Histone code


Readers-

Chromodomain•CH3(Methyl)- recognition domain•HP1 has a chromodomain•Targets to Me-lys or H3K9me•Promote packed “CLOSED” chromatin

Demethylation of Lys 9 in H3 tailfacilitates phosphorylation (P) of Ser 10Acetylation (Ac) of Lys 9 and 14leads to “OPEN” chromatin

BromodomainBinds to acetylated lysines “OPEN”

Wide range of histone modifications >>>

Writers: enzymes that add a mark

Readers: proteins that bind to and “interpret” the mark

Erasers: enzymes that remove a mark

Tarakhovsky, A., Nature Immunology, 2010.

03/11/2016

Reading / translating the histone code


The core histones are among the most conserved eukaryotic proteins; therefore, the nucleosomes formed by these proteins are very similar in all eukaryotes. But there are numerous histone variants found in eukaryotic cells.

Such unorthodox histones can replace one of the four standard histones to form alternate nucleosomes and may serve to demarcate particular regions of chromosomes

Examples-

Moggs and Orphanides, oxicological Sciences, 2004.

Play role in DNA damage repair

When chromosomal DNA is broken, H2A.X located adjacent to the break is phosphorylated at a serine residue that is not present in H2A.

PhosphorylatedH2A.X is specifically recognized by DNA repair enzymes leading to their localization at the site of DNA damage.

03/11/2016

Histone Varients

1) H2A.X is a variant of H2A


In the centromeric region of chromosome , CENP-A replaces the histone H3 subunits in nucleosomes.

These nucleosomes are incorporated into the kinetochore that mediates attachment of the chromosome to the mitotic spindle core structure of the nucleosome.

Consistent with this interaction being critical for kinetochore formation, loss of CENP-A interferes with the association of kinetochore components with centromeric DNA.

Alteration of chromatin by incorporation of histone variants. incorporation of CENP-A in place of histone H3 is proposed to act as a binding site for one or more protein components of the kinetochore. Mol. Bio of Gene, Watson et al., 7th edition

03/11/2016

2) CENP-A varient histone H3


The decreased HAT activity of CBP is a key contributor to the RSTS phenotype.

Features- Broad thumbs and toes, facial abnormalities, congenital heart defects, and increased risk of tumor formation.

Frequency- This condition is uncommon; it occurs in an estimated 1 in 100,000 to 125,000 newborns.

Additional features of the disorder can include eye abnormalities, heart and kidney defects, dental problems, and obesity.

Infants born with this severe form of the disorder usually survive only into early childhood.

RSTS - Facial features (A), left hand and feet showing broad thumb and big toes (B, C) and X-ray of both hands showing short broad thumbs (D). (Limb Malformations & Skeletal Dysplasia)

Huda Y. Zoghbi and Arthur L. Beaudet Cold Spring Harbor Laboratory Press, 2016

Mutations in the CREBBP gene, the gene provides instructions for making CREB binding protein, plays an essential role in controlling cell growth and division and prompting cells to mature

This mutation abolishes the histone acetyltransferase (HAT) activity of CBEP (Murata et al. 2001).

03/11/2016

Diseses associated with histone modification

1. Rubinstein–Taybi Syndrome (RSTS)


The syndrome is caused by mutations in the RPS6KA3 gene (histone phosphorylation) and is located on the short arm of the X chromosome. Males are usually more severely affected than females.

Ribosomal protein S6 kinase, 90kDa, polypeptide 3, also known as RPS6KA3, is an enzyme that in humans is encoded by the RPS6KA3 gene.

The protein RSK2 which is encoded by the RPS6KA3 gene is a kinase which phosphorylates some substrates like CREB and histone H3.

A rare genetic disorder characterized by mental retardation and abnormalities of the head and facial and other areas.

Cardiac abnormalities affect 15% of the patients.

Coffin-Lowry syndrome 03/11/2016

2. Coffin-Lowry syndrome

CLSF- Coffin-Lowry Syndrome Foundation

Created in 1991 by Mary Hoffman in France.

CLS a general forum in which to exchange information, ideas and advice and a great resource for families affected by CLS.

Huda Y. Zoghbi and Arthur L. Beaudet Cold Spring Harbor Laboratory Press, 2016

Department of Plant Biotechnology 3203/11/2016

Techniques to Study Histone Modification

2) Mass Spectrometry (MS)

Other genome-wide techniques combined with ChIPs


ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it.

Chromatin immunoprecipitation (ChIP) has become the technique of choice to investigate protein–DNA interactions inside the cell.

ChIP has been used for mapping the localization of post-translationally modified histones and histone variants in the genome, and for mapping DNA target sites for transcription factors and other chromosome-associated proteins.

There are mainly two types of ChIP, primarily differing in the starting chromatin preparation.

Philippe Collas, Mol Biotechnol (2010) 45:87–100Thomas A. Methods in Molecular Biology, vol. 538

1. Cross-linked ChIP (XChIP)

Uses reversibly cross-linked chromatin as a starting material then sheared by sonication.

Mainly suited for mapping the DNA target of transcription factors or other chromatin-associated proteins,

2. Native ChIP (NChIP)

Chromatin sheared by micrococcal nuclease digestion.

Mainly suited for mapping the DNA

target of histone modifiers.

03/11/2016

1. Chromatin Immunoprecipitation (ChIP)


1. DNA and associated proteins reversibly cross-linked with formaldehyde followed by cell lysis.

2. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion.

3. Immunoprecipitation (IP) by using an appropriate protein-specific antibody.

4. Reverse cross linking and sequence determination

5. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo.

Chromatin immunoprecipitation assay (ChIP) and various methods of analysis

Philippe Collas, Mol Biotechnol (2010) 45:87–10003/11/2016

Major Steps Involved in ChIP Assay


Chromatin Immunoprecipitation (ChIP) – Seq Assay


ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin immunoprecipitaion ('ChIP') with DNA microarray ("chip").

Development of genomic DNA microarrays (chips) has, when combined with ChIP assays, enabled the mapping of transcription factor binding sites and of histone modifications.

The most prominent representatives of this class are transcription factors, replication -related proteins, like Origin Recognition Complex Protein (ORC), histones, their variants, and histone modifications.

Philippe Collas, Mol Biotechnol (2010) 45:87–10003/11/2016

ii) ChIP–on-chip


Chromatin immunoprecipitation followed by sequencing (ChIP–seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes.

1. Crosslinking

2. Chromatin is sheared by sonication (200–600 bp )

3. Immunoprecipitation

4. Reverse crosslinking DNA is assayed to determine the sequences bound by the protein.

5. Construction of a sequencing library,

6. Sequencing of DNA.

Overview of a ChiP–seq experiment.NATuRe RevIewS | GeneticsPeter J. Park 200903/11/2016

ii) ChIP–seq

Major Steps

In ChIP–seq, the DNA fragments of interest are sequenced directly instead of being hybridized on an array.


Illumina Sequencing


Chromatin Immunoprecipitation (ChIP) coupled with quantitative PCR can be used to investigate protein-DNA interaction at known genomic binding sites.

This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.

1. Cell fixation (cross-linking)

2. Chromatin shearing: by Sonication (100-500 bp)

3. Chromatin IP: Using specific ChIP-grade antibodies

4. Reverse cross-linking and DNA purification.

5. qPCR and analysis

EpiTect® ChIP PCR System03/11/2016

Major Steps

iii) ChIP-qPCR


Revolution in Immunoassays


Mass Spectrometry is an analytic technique that utilizes the degree of deflection of charged particles by a magnetic field to find the relative masses of molecular ions and fragments.

A mass spectrum is a plot of the ion signal as a function of the mass-to-charge ratio (m/z)

The two primary methods for ionization of whole proteins,

i). Electrospray ionization(ESI)

ii). Matrix-assisted laser desorption/ionization (MALDI).

03/11/2016

2. Mass Spectrometry (MS)


1.Extraction of histones.

2. Further separation by SDS-PAGE

3. Protease digestion

4. Trypsin is used to cleave proteins

5. Reversed phase chromatography.

6. Mass spectrometer analysis

Quantification and identification of the example peptide H3 27-40 by mass spectrometry

A. The areas of peptides with different modification states are highlighted in grey. The presence of fragmentation spectra for the monomethylated peptide is also displayed.

B. MS/MS spectrum -fragment ions b2+ and b3+ clearly demonstrate methylation in K27 while y6+-y9+ show only propionylation in positions K36 and K37.

03/11/2016

Major Steps

Ignasi Forné et.al., CIPSM,2012


Recent Review on Histone Modification

A genetically encoded probe for live-cell imaging of H4K20 monomethylation


H4K20me1-Mintbody : A new probe to track histone modifications in living cells

Modification specific intracellular antibody

Genetically encoded single chain variable fragment of antibody (scFv) tagged with green fluorescent protein (GFP) recognizes and binds to histone H4 post-translationally modified at K20 position (H4K20me1).

Scientists at Tokyo Institute of Technology have developed a sensitive fluorescent antibody probe(Mintbody) that specifically detects monomethylation of lysine 20 in histone H4 in living cells.

To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells.

© 2016 The Author(s). Published by Elsevier Ltd.

03/11/2016

Mintbody

Two mintbodies have been developed to date1.H3K9Ac2.H3K20me1

Yuko Sato, et. al., JMB, 2016


(a) Schematic structure of H4K20-mintbody.

(b)The localization of H4K20me1-mintbodies in living cells. H4K20me1-mintbody was concentrated in nuclei during interphase and bound to chromosomes during mitosis.

VH- Blue colour, LH- Red colour, The amino acids that differ in 12C8 are indicated in yellow. .Space filling models of two important residues (I95 and A99; letters shown in red) are blown-up to the side. The oxygen and nitrogen atoms arecolored in red and dark blue. Bars, 10 μm.

(c) The crystal structure of 15F11-scFv at 1.94 Å resolution.



Conventional techniques used to study regulation by histone modifications are limited to fixed (dead) cells, thus preventing assessment of histone modification in single, living cells. e.g. ChIP-chip, ChIP-seq , ChIP-qPCR etc.

The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammaliancells in which this target modification was diminished.

In a roundworm Caenorhabditis elegans model, the H4K20me1-mintbody could be used to monitor changes in H4K20me1 over the cell cycle and localization of dosage-compensated X chromosomes without disrupting cell function.

Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle.

This research also identified key amino acids responsible for H4K20me1-mintbody conformational stability, solubility, and consequently, functional performance using X-ray crystallography and genetic analyses.

This research has future implications and can be used to monitor the dynamics of histone modifications and genome integrity in single living cells without disturbing cellular functions.

In the future, development of additional mintbodies specific to diverse post-translational histone modifications will facilitate the identification of regulatory mechanisms that control epigenetic modifications.

03/11/2016

Importance



Histone proteins are most important for packaging and ordering of DNA. N-terminal tails of histones are the most accessible for post translational modificatins. Histone tail modification participate in regulation of many processes including,

– Transcription activation– Transcription repression– DNA damage and repair– Chromatin assembly – Domain bindings– Cell cycle – Long-range packaging (heterochromatin formation, silencing)– Chromosome condensation

Post-translational modification (PTM) of histones play an impotanat role in epigenetic regulation of a gene.

Strategy for localizing histone marks- ChIP-chip, ChIP –seq., ChIP-qPCR, Mass Spectromery etc.

The H4K20me1-Mintbody could be used to monitor changes in H4K20me1 in living cells.

03/11/2016

Conclusion


References

Sato, Y., Kujirai, T., Arai, R., Asakawa, H., Ohtsuki, C., Horikoshi, N., Yamagata, K., Ueda, J., Nagase, T., Haraguchi, T., Hiraoka, Y., Kimura, A., Kurumizaka, H. & Kimura, H., A genetically encoded probe for livecell imaging of H4K20 monomethylation, Journal of Molecular Biology (2016).

Mechanisms of Histone Modifications, Zdenko Herceg and Rabih Murr (download.bioon.com.cn/upload/201105/30165853_5528.pdf )

Shelley L Berger , Histone modifications in transcriptional regulation. Current Opinion in Genetics & Development 2002, 12:142–148.

Sascha Beneke Regulation of chromatin structure by poly(ADP-ribosyl)ation . www.frontiersin.org REVIEW ARTICLE September2012|Volume3|Article169 |

Peter J. Park ChIP–seq: advantages and challenges of a maturing technologyNATUREReviews | Genetics vOlume 10 | OCTOBER 2009 |

Anna Sawicka, Christian Seiser. Histone H3 phosphorylation e A versatile chromatin modification for different occasions. Biochimie 94 (2012) 2193e2201

Eric J. Richards and Sarah C.R. Elgin Epigenetic Codes for Review Heterochromatin Formation and Silencing: Rounding up the Usual Suspects. Cell, Vol. 108, 489–500, February 22, 2002,


EpiTect® ChIP PCR System For reliable analysis of protein—DNA interactions (www.sabiosciences.com/manuals/1073038_PP_GEF_Chip_0712_lr.pdf )

Huda Y. Zoghbi1,2 and Arthur L. Beaudet. Epigenetics and Human Disease Published by Cold Spring Harbor Laboratory Press at University of Hong Kong Libraries on February 1, 2016

Vikki M. Weake and Jerry L. Workman, Histone Ubiquitination: Triggering Gene Activity , Molecular Cell 29, March 28, 2008

Mol Biology of the Gene by Watson 7th Edition

Principles of Biochemistry by Lehninger 4th Edition

https://en.wikipedia.org/wiki/Histone http://www.abcam.com/epigenetics/histone-modifications-a-guide https://www.youtube.com/watch?v=fCd6B5HRaZ8 https://www.youtube.com/watch?v=4oFdS9EN9Pk https://www.youtube.com/watch?v=eYrQ0EhVCYA

Department of Plant Biotechnology 5003/11/2016