Local and Global Effect of Chromatin

Reading from Molecular Biology of the Cell 5th edition

Pages 202-245

Chromosomes- Structure composed of a very long DNA molecule and associated proteins that carries the genetic (and epigenetic) information

- Especially evident in plant and animal cells undergoing mitosis or meiosis, where each chromosome becomes condensed into a compact rod-like structure that is visible by light microscopy

Chromatin- Complex of DNA, histones and non-histone proteins that collectively make up chromosomes

- DNA, histones and non-histone proteins are subject to post-translational/replication modifications that form the basis of an epigenetic code

Chromosomes and Chromatin

A. Interphase chromatin B. A replicated chromosome at mitosis

Compaction of chromatin is cell-stage dependent

Big, basic question: What is the relationship between structure of chromatin and gene expression ?

Most interphase chromatin is condensed into 30nm coils.

Interphase chromatin ~500 fold compaction end-to-end

Mitotic chromatin 20X fold compaction end-to-end over interphase

levels of Chromatin compaction

A) 30 nm fibersB) beads on a string-nucleosome from interphase nucleus

The nucleosome is a basic unit of chromatin 1974

Nucleosome = a nucleosome core particle + linker DNA+ a linker histone

DNA length: 180-200 bp

Nucleosome core particle = histone octamer + 146 bp DNA

Nomenclature

Histones- highly basic (+) proteins

Protein Molecular weight

Major basic Amino acids

H1 21 Lys++

H2a 13.8 Lys

H2b 13.8 Lys

H3 15.4 Arg/Lys

H4 11.4 Arg/Lys

Histone fold-3 alpha helices and 2 folds

Nucleosome core particle

2.8 A crystal structure of theMono-nucleosome

1. DNA (146 bp) is wrapped in 1.75 left-handed superhelical turns

per nucleosome

2. One side of DNA is in contact with histone octamer, the other is

solvent exposed

3. DNA helical turns in a nucleosome have an average of 10.2 bases

per helical turn versus 10.5 for DNA in solution

How is nucleosomal DNA different from free DNA

Translational positioning Rotational Positioning

linker

Core particle

Nucleosome Positioning

Transcription factor binding to DNA is inhibited within nucleosomes

• Chromatin assembly inhibits transcription by all three RNA polymerases in vitro.

• In vivo genetic evidence links histones to repression (and activation as well).

• Affinity of transcription factors for its cognate DNA binding site is decreased when DNA is reconstituted into nucleosomes.

• The binding of TBP to the TATA box is very sensitive to chromatin assembly in vitro.

• Extent of inhibition is dependent on:

– Location of the binding site within the nucleosome

• binding sites at the edge are more accessible than the center

– The type of DNA binding domain

• Zn finger transcription factors bind to nucleosomal DNA more easily than bHLH domains.

ArticleNature 442, 772-778 (17 August 2006)

A genomic code for nucleosome positioning

Eran Segal et al (Jonathan Widom)

Abstract

Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins. Nucleosomes have higher affinity for particular DNA sequences, reflecting the ability of the sequence to bend sharply, as required by the nucleosome structure. However, it is not known whether these sequence preferences have a significant influence on nucleosome position in vivo, and thus regulate the access of other proteins to DNA. Here we isolated nucleosome-bound sequences at high resolution from yeast and used these sequences in a new computational approach to construct and validate experimentally a nucleosome–DNA interaction model, and to predict the genome-wide organization of nucleosomes. Our results demonstrate that genomes encode an intrinsic nucleosome organization and that this intrinsic organization can explain approx. 50% of the in vivo nucleosome positions. This nucleosome positioning code may facilitate specific chromosome functions including transcription factor binding, transcription initiation, and even remodelling of the nucleosomes themselves.

Naked DNA

Chromatin

Lev

el o

f ac

cess

ibil

ity/

Tra

nsc

rip

tion

al a

ctiv

ity

Unavoidably High levels ofTranscription

Mechanisms for increased accessibility or activationMechanisms for

decreased accessibility or repression

High

low

How to actively transcribe a gene embedded within repressive

chromatin?

• Cooperative binding of multiple factors.

• Utilize various chromatin remodeling activities to make chromatin compatible for transcription

TATA

–Modulation by incorporation of histone variants

–Modulation by ATP-driven chromatin remodeling complexes

–Modulation by enzymes that post-translationally modify

histonesAcetylationMethylationUbiquitinationPhosphorylationADP-ribosylation

Histone association with DNA is dynamic ‘on its own’ 4 second average residencyDNA is exposed for 10 to 50 milliseconds

prior to reassociation

Mechanisms for chromatin remodeling

Modulation by Histone Variants• In addition to the major histones H2A, H2B, H3 and H4, many

organisms also have distinct batteries of histone variants– H2AZ, H2AX and macroH2A H2AZ has been shown to associate with actively transcribed chromatin

regions H2AX has been shown to be crucial for chromatin decompaction during

DNA repair. Phosphorylation on its SQE/DØ sequence is one of the earliest events in response to double-strand DNA breaks

– H3.3 and CenH3s (CenpA in human) H3.3 is a replacement H3 variant CenH3s are centromere-specific H3 variants

• Histones H2B and H4 have very few variants

The chromatin structure and function can be different dependent on the presence or absence of histone variants which possess different amino acid sequences.

• Yeast SWI/SNF– 10 proteins

– Needed for expression of genes involved in mating-type switching and sucrose metabolism (sucrose non-fermenting).

– Some suppressors of swi or snf mutants are mutations in genes encoding histones.

– Interacts with chromatin to activate a subset of yeast genes.

– Is an ATPase-containing complex

• Mammalian homologs: hSWI/SNF– The ATPase component is BRG1, related to Drosophila

Brahma

• Other ATP-dependent chromatin remodeling complexes have been discovered

Remodeling by ATP-driven chromatin remodeling complexes

Involvement of SWI/SNF in transcription

• The SWI/SNF complex is required for transcriptional activation of 5% yeast genes.

Can be recruited directly through interaction with DNA binding transcription factors.

Can be recruited indirectly by interaction with other transcriptional coactivators or along with the RNA polymerase holoenzyme.

SWI/SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition.

Dependent on the chromatin organization as well as the transcription factors involved, the SWI/SNF also contributes to transcriptional repression.

How do chromatin remodeling complexes work?

• Structural alteration

• Nucleosome sliding

• Nucleosome eviction

The consequence of chromatin remodeling is dependent on the type of chromatin

remodeling complexes involved

A common route involves ATP hydrolysis to do the following:

Nucleosome sliding induced by ISWI-containing complexes

• Make nucleosome mobile in the presence of ATP

• Also involved in nucleosome/chromatin assembly

• Has roles in both transcriptional activation and repression (occlude exposed

transcription factor binding site

NURF +ATP

Move nucleosome from its

stable, low energystate position

NURF +ATP

TF

NURF +ATP

Evenly spacenucleosomes thatwould otherwiseclump due toinfluence of DNAsequence

Expose transcription Factor binding site

Chromatin remodeling by covalent modification of

histones

Multiple modifications provide for an enormous number of potential combinations

Histone Acetyltransferases (HATs)

• Type A nuclear HATs: acetylate histones in chromatin.

• Type B cytoplasmic HATs: acetylate free histones prior to their assembly into chromatin.– Acetylate K5 and K12 in histone H4

Use acetyl-coA as donor coA

Highly acetylated histones are associated with actively transcribed chromatin– Increasing histone acetylation can turn on some

genes– Chromatin immunoprecipitation (ChIP) of DNA with

antibodies against Ac-histones pulls down actively transcribed genes

– The acetylated chromatin is more “open”• Increased DNase sensitivity• Increased accessibility to transcription factors

and polymerases

Some common coactivators are nuclear HATs

• Gcn5p is a yeast transcriptional activator of many genes in yeast that possesses HAT activity

• PCAF (p300/CBP associated factor) is the human homolog of yeast Gcn5p

• p300 and CBP are similar HATs that interact with many transcription factors (e.g. CREB, AP1 and MyoD)

• p300/CBP are needed for activation by many transcription factors, and thus are considered as general coactivators or

cointegrators

• p300/CBP has intrinsic HAT activity as well as binding to the HAT PCAF

Roles of histone acetylation

• To increase access of transcription factors to DNA in nucleosomes.

• To decondense 30nm chromatin fibers• To serve as epigenetic marks for binding of

non-histone proteins (e.g. bromodomain proteins like Gcn5p/CBP/p300/PCAF) to chromatin

Histone deacetylation is catalyzed by histone deacetylases and associated with

transcriptional repression

Histone deacetylases (HDACs):1. Three classes, about 20 identified members2. can be recruited by transcriptional repressors to

specific target genes and/or deacetylate histones in chromatin in a non-targeting, global fashion

3. Acetylation and deacetylation are very dynamic events

4. Aberrant histone deacetylation has been linked to cancer

Mammalian HDACs have been classified into three classes

Class I (HDACs 1, 2, 3 & 8) homologs of yeast RPD3 and localize to the nucleus.

Class II (HDACs 4, 5, 6, 7, 9 & 10) are homologs of yeast Hda1 and are found in both the nucleus and cytoplasm.

Class III (Sirt1 - Sirt7) are homologs of yeast Sir2 and form a structurally distinct class of NAD-dependent enzymes found in both the nucleus and cytoplasm. 

Information about three classes of HDACs

• Class I HDACs are relatively small in size, abundant, ubiquitously expressed, mainly nuclear, sensitive to TSA and tend to associate with corepressor proteins to form large corepressor complexes.

• Class II HDACs are relatively larger in size, less abundant, shuffle between cytoplasm and nuclei, likely tissue-specific and sensitive to TSA.

• Class III HDACs are involved in silencing of rRNA genes , telomere silencing and polII-transcribed genes and not sensitive to TSA. They require NAD as a cofactor.

Histone Methylation• Two chemical classes of HMTs: arginine specific-HMTs

and lysine-specific HMTs.• Both Arg and Lys can be mono-, di- or tri-methylated.• In contrast to histone acetylation, histone methylation is

believed to be stable until recently. This modification is still rather stable though. Turnover rate of histone methylation is similar to that of histone turnover

• Methylation does not neutralize positive charge on Lys and Arg.

• Methyl group is thought to be too small to have a direct effect on chromatin structure.

• The function of histone methylation is believed to be mediated through specific methylated histone binding proteins (as part of histone code hypothesis).

Approaches for identification and characterization of HMTs

1. Biochemical purification of HMT activities using in vitro HMT assay.

2. Sequence similarity: testing the proteins containing a SET domain.

The first identified Arg-specific HMT is Carm1/PRMT4

The first identified Lys-specific HMT is SUV39h1 based on its similarity to a plant protein methyltransferase. The HMT activity resides in the SET domain

HMTase Histones Sites

ySET2 H3 K36

G9a H3 K9

CARM1 H3 R2, R17, R26

PRMT1 H4 R3

Summary of Known HMTases and Their Target Sites

ySET1/mSET7 H3 K4

SUV39H1/Clr4/ESET H3 K9

DOT1 (no SET) H3 K79

SET8 H4 K20

Roles in transcription

elongation

silencing

activation

activation and elongation*

activation and elongation*

silencing/X-inactivation

activation

silencing, cell cycle

EZH1/EZH2 H3 K27 silencing/X-inactivation

How histone methylation affects chromatin function?

• H4-R3 methylation facilitates acetylation of H4 by p300 (why it is associated with activation.

• H3-K9 methylation creates a binding site for HP1 and HP1 is known to associate with HDACs and involved in heterochromatin formation (why it is involved in repression)

• H3-K4 methylation prevents binding of NuRD• The underlining mechanism for many other

modifications is not clear

Reversibility of histone methylation

Non-specific histone demethylation1. Histone tail clipping by an endoprotease.2. Histone replacement by unmodified histones or histone

variants.

Specific demethylation 1. Arginine demethylase: PAD4 (peptidylarginine

deiminase 4) 2. The amine oxidase family lysine demethylases LSD1

(lysine specific demethylase 1) H3K4Me and H3K9Me 3. The jmjc family demethylases

JHDM1: H3K36Me2JHDM2A and JHDM2B: H3K9Me2JHDM3A/JMJD2A: H3K9Me3/2 and H3K36Me3/2

Histone phosphorylation• Phosphorylation on Ser-10 of H3 is involved in

both transcriptional activation and in chromosome condensation during mitosis

• Phosphorylation on Ser-10 of H3 may facilitate acetylation of histone H3 on Lys-9 and Lys-14

• Phosphorylation of histone H2AX variant is involved in DNA repair

• Phosphorylation of Ser14 of H2B is linked to apoptosis

• Many Ser and Thr sites in histone tails can be phosphorylated. In most cases the functional consequence is not clear

Histone Ubiquitination• H2A (Lys-119) and H2B (Lys-123) can be mono-

ubiquitinated• Mono-ubiquitination is not associated with protein

degradation by the proteasome• H2B ubiquitination in yeast is catalyzed by Rad6

and is required for methylation on Lys-4 and Lys-79 of H3 (trans-histone effect). The underlining mechanism is unknown.

• Both ubiquitination and de-ubiquitination are involved in transcriptional regulation (Genes and Development 17: 2648-2663, 2003)

5 8 12 163Ac-N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...Human H4

20

Me MeAc Ac Ac Ac

Human H3 N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...Ac Ac Ac AcMe MeMe

4 9 14 18 23 27

p pMe MeMe

Me

Interplay Between Different Histone Modifications

1. At the level of modification2. At the level of function

P-S10 inhibits binding of HP1 to H3K9(Me)2/3.Fischle et al., Nature. 2005 Dec 22;438(7071):1116-22

1stPRMT1

2nd

p300

The ‘Histone Code’

Histone code hypothesis: that multiple histone modifications, acting in combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions.

How the histone code be read? Likely read by specific protein domains

Code Protein Motif

Ac-Lys Bromo – on SWI/SNF proteins

H3K9Me HP1 Chromo

H3K27Me Polycomb Chromo

Phos-H3 S10 ? Cell cycle/mitosis

Different combinations ?

H3K4Me WDR5 – Needed for Hox gene activation

The functions of SWI/SNF and the SAGA complex are genetically

linked• Some genes require both complexes for activation.• Other genes require one or the other complex.• Many genes require neither – presumably they may utilize

different ATP-dependent complexes and/or HATs

Functional interplay among different chromatin remodeling factors

Chromatin Structure and FunctionGlobal Level

Interphase Nucleus: euchromatin vs heterochromatin

46

Interphase chromatin exists in two general states

A. Euchromatin - Less-condensed state – open, dispersed and potentially active in transcription– often located near nuclear pores– acetylated histones, H3-K4 methylation, and low DNA

methylationB. Heterochromatin – more-condensed state

– Usually located at the periphery of the nucleus– this DNA in general is not transcribed– Hypoacetylated histones, low H3-K4 methylation,

methylation at H3-K9, H3-K27 and H4-K20, and DNA methylation

22.228 lecutre 7 47

Constitutive vs facultative heterochromatin1. Constitutive heterochromatin, condensed at all times

– the centromere– the telomeres

2. Facultative heterochromatin, transient condensation, contains potentially active genes

– Inactive X chromosome known as the “Barr body”– facultative heterochromatin becomes more abundant in

cells as the organism matures from embryo to adult and aging, as cells specialize, and gene expression is restricted

Epigenetics

Epigenetics is the study of heritable mechanisms that affect the transcriptional state of a gene which is not due to change in DNA sequence

Molecular mechanisms that mediate epigenetic phenomena include (but is not limited to): DNA methylation, histone modifications and RNAi machinery (specific chromatin structures that allow stable transcriptional activation or silencing)

Position-effect variegation (PEV): euchromatic genes become subject to transcriptional silencing as a result of their placement adjacent to heterochromatin by chromosomal rearrangements. Gene silencing by heterochromatisation in PEV is clonally initiated in a variable number of cells resulting in the variegated phenotype.

Su(var) : genes identified in Drosophila genetic screens that contribute positively to heterochromatin formation. HP1, Su(var)3-9

E(var) : genes identified in Drosophila genetic screens that contribute negatively to heterochromatin formation Herman Muller 1938

Figure 9-65. Position-effect variegation in Drosophila. (A) Heterochromatin (red) is normally prevented from spreading into adjacent regions of euchromatin (green) by special barrier sequences of unknown nature. In flies that inherit certain chromosomal translocations, however, this barrier is no longer present. (B) During the early development of such flies, the heterochromatin now spreads into neighboring chromosomal DNA, proceeding for different distances in different cells. The spreading soon stops, but the established pattern of heterochromatin is inherited, so that large clones of progeny cells are produced that have the same genes condensed into heterochromatin and thereby inactivated (hence the "variegated" appearance of some of these flies; see Figure 9-51B). This phenomenon shares many features with X-chromosome inactivation in mammals.

H3K9 methylase

Positive feedback and heterochromatin spreading

Insulator/Boundary element: elements that modulate interactions between other cis-acting sequences and separate chromatin domains with distinct condensation states. Thus, they are proposed to play an important role in the partitioning of the genome into discrete realms of expression.

Chicken -globin domain

Insulator InsulatorCTCF binding site

Imprinting: Genomic imprinting describes the preferential or exclusive expression of a gene from only one of the two parental alleles. The allele-specific expression of imprinted genes is based on allele-specific epigenetic modifications such as cytosine methylation and histone acetylation and methylation.

Imprinting Control Region

methylation

Read the paper that describeshow parental imprinting isrelated to GWAS studiesfor specific risk allelesingle nucleotide polymorphisms(SNPs)

Chromatin domain: The organization of one or more genes into an i) expressed, ii) potentially expressed, or iii) silenced region defined by insulators and/or sites of attachment to the nuclear matrix (The activity state is often related to covalent modification of histones and/or DNA, in addition to the association of specific regulatory proteins).

i) Insulator/boundary element

ii) Scaffold attachment regions (SARs)

iii) Matrix attachment regions (MARs)

Mapping higher orderChromatin interactions

Living cell

Formalin-Cross linkedchromatin

Ligate, thenReverse crosslinks and sequence

Estrogen receptor- regulated genes form chromatin loops

Loop geneanchor gene anchor gene

ChIA-PET clones

ERIP

For the ER ChIA-PET analysis:

Long range interactions are intrachromosomal

Loops vary between 100 kb to 1 Mb in distance

Stronger ER binding sites correlate with duplex/complexER chromatin loops

Anchor genes near interacting ER binding site loopsare expressed more strongly than genes near non-interacting ER binding sites

Dosage Compensation

Males (XY) and females (XX) must generate equal amounts of most X-linked gene products

Three distinct mechanisms:Mammals: one of X chromosome is inactivated

D. melanogaster: Males double transcription from the single X chromosome to equal XX females

C. elegans: Females reduce transcription from both XX by half to equal XO males

Dosage Compensation and X-chromosome Inactivation

In mammals, Xist RNA is transcribed from the X inactivation center (XIC) and spreads to inactivate the X chromosome in cis. Xist transgenes inserted on autosomes also cause the spreading of transcriptional inactivation in cis.

In flies, there are about 35 chromatin entry sites (CES) on the X chromosome, including roX1 and roX2. Unlike the XIC in mammals, the entry sites in flies regulate twofold transcriptional activation.

1.7 kb ncRNA

Epigenomics - genome-wide study of epigenetic features on DNA

Epigenetics - refers to heritable changes in phenotype (appearance) or gene expression caused by mechanismsother than changes in the underlying DNA sequence

Omics - referring to totality of some sort

In the past: Studies of relationship between epigenetic marks on chromatin and gene expression focused on a single gene

Now/near future: Relationship between chromatin and gene expressioncan be understood across the genome

What new rules, patterns or statistics emerge from this effort?

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project Nature. 2007 June 14; 447(7146): 799–816. The ENCODE Project Consortium

200 experiments; 30 Mbases of DNA; 400 million data points

How do we know that non-coding DNA isn’t junk DNA?

Conservation analysis:

~5% of human genome under purifying selection pressure based on all metazoan genomes

~20% of human and mouse genomes subject to purifying selection pressure

Only 1.2% of the human genome encodes for proteins (exons)

What is the significance of non-protein coding DNA?

Epigenomic studies might provide an answer to this question.

Genomics - Primary DNA sequence - Evolutionary conservation of primary sequence - Shared synteny - positional co-localization of genes on chromosomes of different species – i.e., Hox cluster - Copy number variations, indels - SNPs – single nucleotide polymorphisms

Epigenomics – genome-wide mapping of: - DNA methylation of the genome - histone post translational modifications - DNA hypersensitive sites - DNA tethering to locus control regions - Transcription factor binding to DNA

Phenotype - GWAS – genome wide association studies (genes/loci associated with disease) - proteomics – spectrum of proteins expressed - manifestation of differentiated cell types and tissues

Transcriptome – transcriptional output – mRNA, miRNA, ncRNA

Disease risk alleles

Personalized genomics is now an affordable reality

Illumina 550k SNP Chip array

Tiling Array – much greaterCoverage than “expression arrays”up to 6 million probes – 545 timesbigger

Transcriptome profiling:

224K or tiling arrays usedto identify transcripts

rather than 11k expressionarrays based on RefSeqgene exons

Lots of transcriptional ‘dark matter’Non coding RNAs

Transcriptome profiling

ChIP-CHIP mapping of the epigenome

Antibodies for acetylated or methylated histones, transcription factors,high mobility group factors, coactivators used for mapping the distributionof corresponding epigenetic features on the genome

Protein of interest

Epigenomic identification of methylated DNA

Bisulfite sequencing of the genome – defines actual sites of DNA methylationbut is more costly

Identification of DNAse hypersenstive sites – open chromatin

Also – Formaldehyde assisted isolation of regulatory elements (FAIRE) is usedisolates open instead of closed DNA

Sample of ENCODE epigenomic anotation

Replicationtiming

DNAse hypersensitivesites

Regulatory factorBinding regions

http://genome.ucsc.edu/ENCODE/encode.hg18.html

Looking at ENCODE data

ES

MEFH3K4me3

A look at multiple histone marks in the mouse genome

H3K4me3 marks at 5’ end of genes

H3K36me3 – marks transcriptional elongationAox1 – expressed strongly in brain

Developmentally poised promoters in ES cells

Activating and repressive marks both present in pluripotentCells – These represent genes with complex stage and tissueSpecific expression patterns

H3K27me3 – repressive mark – in differentiated cells it has theSame pattern as H3K9Me

H3K36me3 – a mark for transcriptional elongation

Differentiation

Proximal

DHS not near TSS

H3K4me1Marks insulatorDNA and enhancers

Transcription factor and methylated histone proximity to transcription start sites (TSSs)

RFBRs – regulatory factor binding regions

repressive mark

Evolutionary constraints in DNA sequence

Open chromatin,Transcription factorBinding and modifiedhistones

Protein coding

Evolutionarily conserved DNA – relationship with coding sequence,exposed chromatin and RFBRs (transcription factors and methylated histones)

Ancient repeatsevo. neutral

contained within feature

Window surrounding feature

Constrained sequences

Codingsequences

Ancient repeats

Open chromatin

Transcription factors

SN

P

Single nucleotide polymorphisms exist more frequently in regulatoryDNA sequences than in coding sequences

- more open to evolutionary change

Intraspecies constraint for small RNAs

This looks at differences between humans only

H3K4me2 and H3K4me3 histone marks are biased towardCoding DNA sequence

Seila et al. Science 322, 1849 (2008)

Cost and effort issues

# of epigenetic marks to look at (DNA methylation, H3K4me2, H3K4me3, H3K9me3, H4K20Me…

100

X

# of tissues and cell lines (NCI 60, others from ATCC, ES cells…)

100

X

# of biological conditions to assess (G1/S/G2/M, hormone treatment, growth factors…)

100 = 1,000,000 experiments

X the cost of a single ChIP-Seq or ChIP-CHIP run

$5000

= 100 x 100 x 100 x $5000 = $5,000,000,000 ($5 billion)

The End