Local and Global Effects of

Chromatin

Reading from Molecular Biology of the Cell 5th edition

Pages 202-245

Chromosomes and
Chromosomes
Chromatin
- 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 posttranslational/replication modifications that form the basis of an
epigenetic code

Compaction of chromatin is cell-stage dependent

A. Interphase chromatin

B. A replicated chromosome
at mitosis

Big, basic question: What is the relationship

between structure of chromatin and gene
expression ?

levels of
Chromatin compaction

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

The nucleosome is a basic unit of chromatin

1974

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

Nomenclature
Nucleosome = a nucleosome core particle + linker
DNA+ a linker histone
DNA length: 180-200 bp
Nucleosome core particle = histone octamer + 146 bp DNA

Figure 4-23 Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-23 (part 1 of 2) Molecular Biology of the Cell ( Garland Science 2008)

Histones- highly basic (+) proteins

Protein

H1

Molecular
weight
21

Major basic
Amino acids
++
Lys

H2a

13.8

Lys

H2b

13.8

Lys

H3

15.4

Arg/Lys

H4

11.4

Arg/Lys

Histone fold3 alpha helices

and 2 folds

Figure 4-26 (part 1 of 2) Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-26 (part 2 of 2) Molecular Biology of the Cell ( Garland Science 2008)

Nucleosome Positioning

linker

Core particle

Translational positioning

Rotational Positioning

Figure 4-27 Molecular Biology of the Cell ( Garland Science 2008)

Nucleosome core particle

2.8 A crystal structure of the

Mono-nucleosome

Figure 4-28 Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-29 Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-30 Molecular Biology of the Cell ( Garland Science 2008)

How is nucleosomal DNA different from free DNA

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

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.

Article
Nature 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 nucleosomeDNA 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

Level of accessibility/
Transcriptional activity

High

low

Unavoidably
High levels of
Transcription

Mechanisms for
decreased
accessibility or
repression

Mechanisms for
increased
accessibility or
activation

Chromatin

How to actively transcribe a gene

embedded within repressive
chromatin?
TATA

Cooperative binding of multiple factors.

Utilize various chromatin remodeling activities to
make chromatin compatible for transcription

Mechanisms for chromatin remodeling

Modulation by incorporation of histone variants

Modulation by ATP-driven chromatin remodeling complexes

Modulation by enzymes that post-translationally modify
histones
Acetylation
Methylation
Ubiquitination
Phosphorylation
ADP-ribosylation
Histone association with DNA is dynamic on its own
4 second average residency
DNA is exposed for 10 to 50 milliseconds prior
to reassociation

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/D 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.

Figure 4-41 Molecular Biology of the Cell ( Garland Science 2008)

Remodeling by ATP-driven
chromatin remodeling complexes

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

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?
A common route involves ATP
hydrolysis to do the following:

Structural alteration
Nucleosome sliding
Nucleosome eviction
The consequence of chromatin remodeling is
dependent on the type of chromatin
remodeling complexes involved

Nucleosome sliding induced by

ISWI-containing complexes
NURF +ATP

NURF +ATP

TF

NURF +ATP

Evenly space
nucleosomes that
would otherwise
clump due to
influence of DNA
sequence

Move nucleosome from

its
Expose transcription
stable, low energy
Factor binding site
state position
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

Chromatin remodeling by
covalent modification of
histones

Multiple modifications provide for an

enormous number of potential combinations

Figure 4-38 Molecular Biology of the Cell ( Garland Science 2008)

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 members
2. 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.

Generation of nested deletions of Nkx2-5-GFP BAC identified the most distal 5 border of the
cardiac regulatory locus.

2005 by National Academy of Sciences

Chi X et al. PNAS 2005;102:13490-13495

Pairwise alignment of the mouse and human Nkx2-5/Csx loci revealed multiple upstream
noncoding regions of high homology.

Pairwise alignment of the mouse and human Nkx2-5/Csx loci revealed multiple upstream
noncoding regions of high homology. Conserved sequences are shown relative to their position
in the mouse (horizontal axes), and their percent cross-homology between mouse and human
sequences (50-100%) are indicated on the vertical axes. The location of the gene texas is
indicated by double underlines.
2005 by National Academy of Sciences

Chi X et al. PNAS 2005;102:13490-13495

Transgenic analysis of the conserved noncoding regions functioned as previously

uncharacterized enhancers for expression in the interventricular septum (IVS), atrial
ventricular canal, and atria in mouse embryos.

2005 by National Academy of Sciences

Chi X et al. PNAS 2005;102:13490-13495

Identification of the tongue enhancer identified by UH4-Hsp68lacZ transgene expression

patterns.

2005 by National Academy of Sciences

Chi X et al. PNAS 2005;102:13490-13495

Histone H4 acetylation patterns correlated well with temporal and spatial activity of Nkx2-5
distal enhancers and the activation of the proximal AR2 and G-S modules during ES cell
induced cardiogenesis.

Histone H4 acetylation patterns correlated well with temporal and spatial activity of Nkx2-5 distal enhancers and
the activation of the proximal AR2 and G-S modules during ES cell induced cardiogenesis. (A) The antiacetylhistone H4 pattern of Nkx2-5 enhancers from embryonic and neonatal tissues. (B) The RT-PCR analysis of
ES cells and embryoid bodies at different time points after aggregation, with and without noggin. (C) The histone
H4 acetylation pattern of AB2.2 ES cells and embryoid bodies at days 6 and 8 of differentiation. pNoggin-CS2+
expression plasmid was transfected into ES cells to block BMP signaling. pCS2+ vector served as a transfection
control. (D) Whole-mount X-gal staining of an E7.5 embryo carrying the G-SHsp68lacZ transgene and a
schematic diagram of the enhancers assayed by ChIP for histone acetylation patterns.
2005 by National Academy of Sciences

Chi X et al. PNAS 2005;102:13490-13495

A general model for murine embryonic Nkx2-5 transcriptional regulation.

Chi X et al. PNAS 2005;102:13490-13495

2005 by National Academy of Sciences

TheHistoneCode
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

H3K4Me
Phos-H3 S10
Different combinations

WDR5 Needed for Hox gene activation

? Cell cycle/mitosis
?

Bromodomain is the first protein module to be shown to selectively interact with a

covalent mark (acetylated lysine) in the histone NH2-terminal tail
Bromodomain is also present in many transcriptional regulators having intrinsic
histone acetyltransferase (HAT) activity (e.g., GCN5, PCAF, TAFII250).
TAFII250, which itself harbors several histone-modifying activities, contains two
tandem copies of the bromodomain. In this configuration it preferentially binds
diacetylated histone peptides presenting acetyl-lysine moieties that are appropriately
spaced.
Chromodomains, on the other hand, appear to be targeting modules for methylation
marks. The chromodomain of HP1 is highly selective for methylated H3 at Lys9, and
little if any binding is observed with H3 peptides containing a methylated Lys4
position.
Su(var)3-9 HMTase family members also contain a chromodomain, whose integrity is
critical for silencing in vivo.
Several repressive chromatin-remodeling complexes comprise components such as
the Mi-2/CHD ATPase subunit of the NuRD complex, which harbors two
chromodomains and might conceivably recognize dimethylated histone tails in a
manner analogous to double bromodomains. Lys9 and Lys27 in the H3 tail are
embedded in similar sequence motifs, and both positions are hot spots for
methylation by the SET domain containing HMTase G9a.

Some evidence is emerging about a possible combinatorial code. For

example, the histone H3 NH2-terminus appears to exist in two distinct
modification states that are likely to be regulated by a switch between
Lys9 methylation and Ser10 phosphorylation
Ser10 phosphorylation inhibits Lys9 methylation but is synergistically
coupled with Lys9 and/or Lys14 acetylation during mitogenic and hormonal
stimulation in mammalian cells. In this phosphorylated-acetylated state, the
modified H3 tail marks transcriptional activation. H3 phosphorylation is also
important for mitotic chromosome condensation, where it may be linked to
other secondary signal(s) such as the nucleosomal incorporation of the
pericentric H3 analog Cenp-A .
Conversely, aberrant Lys9 methylation antagonizes Ser10 phosphorylation,
leading to mitotic chromosome dysfunction. Further, deacetylation of Lys14
in H3 is required to facilitate subsequent Lys9 methylation by the Clr4
HMTase, again highlighting an ordered interplay to establish distinct histone
tail modifications.
Although the single H3-Lys9 methyl epitope appears sufficient to recruit
HP1 to heterochromatic regions, acetylation of Lys12 in H4 is another
repressive Mark that may help to reinforce a silent chromatin state.

Fig. 2. Translating the histone code. (A) Described protein

modules of histone-modifying enzymes that have been shown to
interact with site-speciic methylation (chromodomain) or acetylation
(bromodomain) marks in histone NH2-termini. A protein module that
would selectively recognize phosphorylated positions is currently not
known. Abbreviations: HMT, histone methyltransferase; HAT,
histone acetyltransferase; HDM, histone demethylase; PPTase,
protein phosphatase; HDAC, histone deacetylase. (B) Proposed
histone tail interactions for a reversed histone code, showing a
chromodomain-containing HAT (e.g., Esa1) and part of a
nucleosome-remodeling complex that may comprise a bromodomaincontaining, inactive HMTase (dashed lettering), such as the trx-G
protein HRX. (C) Possible functional interactions between
Su(var) and Pc-G proteins or between histone- and DNAmethylating enzymes that could be induced or stabilized by siteselective combinations of methylation marks.

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).

Figure 4-38a Molecular Biology of the Cell ( Garland Science 2008)

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

Figure 4-39b Molecular Biology of the Cell ( Garland Science 2008)

SummaryofKnownHMTasesandTheirTargetSites
HMTase

Histones

Sites
R2, R17, R26

Roles in transcription

CARM1

H3

activation

PRMT1

H4

R3

activation and elongation*

SUV39H1/Clr4/ESET H3

K9

silencing/X-inactivation

ySET1/mSET7

H3

K4

activation and elongation*

ySET2

H3

K36

elongation

G9a

H3

K9

silencing

EZH1/EZH2

H3

K27

silencing/X-inactivation

DOT1 (no SET)

H3

K79

activation

SET8

H4

K20

silencing, cell cycle

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 demethylation
1. 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: H3K36Me2
JHDM2A and JHDM2B: H3K9Me2
JHDM3A/JMJD2A: H3K9Me3/2 and H3K36Me3/2

Figure 4-42 Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-43 Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-44a Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-44b Molecular Biology of the Cell ( Garland Science 2008)

Figure 4-45 Molecular Biology of the Cell ( Garland Science 2008)

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 monoubiquitinated
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 Lys79 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)

InterplayBetweenDifferentHistoneModifications
Human H3

Me Me

Me
Ac p

Ac

MeAc

Ac

Me Me p

14

18

23

27

NARTKQTARKSTGGKAPRKQLATKAARKSAP...
4

Me

2nd
Human H4

Me Ac

Ac

Ac

Ac

Me

3 5

12

16

20

AcNSGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...

PRMT1

1st

p300

1. At the level of modification

2. 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

Functional interplay among different

chromatin remodeling factors

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

Chromatin Structure and Function

Global Level

Interphase Nucleus: euchromatin vs

heterochromatin

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
methylation
B. 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
75

Constitutive vs facultative heterochromatin

1. 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

22.228 lecutre 7

76

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.

Positive feedback and heterochromatin spreading

H3K9 methylase

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.

CTCF
This gene is a member of the BORIS + CTCF gene family
and encodes a transcriptional regulator protein with 11 highly
conserved zinc finger (ZF) domains. This nuclear protein is
able to use different combinations of the ZF domains to bind
different DNA target sequences and proteins. Depending
upon the context of the site, the protein can bind a histone
acetyltransferase (HAT)-containing complex and function as
a transcriptional activator or bind a histone deacetylase
(HDAC)-containing complex and function as a transcriptional
repressor. If the protein is bound to a transcriptional insulator
element, it can block communication between enhancers
and upstream promoters, thereby regulating imprinted
expression. Mutations in this gene have been associated
with invasive breast cancers, prostate cancers, and Wilms'
tumors. Alternatively spliced transcript variants encoding
different isoforms have been found for this gene.

Chicken -globin domain

Insulator

CTCF binding site

Insulator

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 describes

how parental imprinting is
related to GWAS studies
for specific risk allele
single nucleotide polymorphisms
(SNPs)

The neighboring Igf2 and H19 genes are expressed monoallelically

from opposite chromosomes and are generally recognized as the
paradigm of mammalian genomic imprinting.
The cis elements underlying their parent-of-origin-dependent
expression patterns were traced to a differentially methylated
region (imprinting control region [ICR]) located 2 kb upstream of the
H19 transcriptional unit. Subsequently, it was documented that
repression of the paternal H19 allele was initiated, but not
maintained by the H19 ICR, while the continuous presence of this
region was required to suppress the maternal Igf2 allele.
The 11-zinc finger protein CTCF, which associates with all known
vertebrate chromatin insulator cis elements, interacted with these
repeat elements in a methylation-sensitive manner. Importantly,
CTCF interacts in vivo with only the maternal H19 ICR allele, which
is consistent with an involvement with the in vivo insulation of the
maternal Igf2 from downstream lineage-specific enhancers.

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)

Living cell

FormalinCross linked
chromatin

Mapping higher order

Chromatin interactions

Ligate, then
Reverse cross
links and sequence

Estrogen receptor- regulated genes form chromatin loops

ER
IP

ChIA-PET clones

anchor gene

Loop gene

anchor gene

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/complex
ER chromatin loops
Anchor genes near interacting ER binding site loops
are 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

1.7 kb ncRNA
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.

Epigenomics -

genome-wide study of epigenetic features on DNA

Epigenetics -

refers to heritable changes in phenotype

(appearance) or gene expression caused by mechanisms
other 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 expression
can 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): 799816.
The ENCODE Project Consortium
200 experiments; 30 Mbases of DNA; 400 million data points

How do we know that non-coding DNA isnt 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

Phenotype
Transcriptome transcriptional
output mRNA, miRNA, ncRNA

- GWAS genome wide association studies (genes/loci

associated with disease)
- proteomics spectrum of proteins expressed
- manifestation of differentiated cell types and tissues

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

Personalized genomics is now an affordable reality

Disease risk alleles

Illumina 550k SNP Chip array

Transcriptome profiling

Tiling Array much greater

Coverage than expression arrays
up to 6 million probes 545 times
bigger

Transcriptome profiling:
224K or tiling arrays used
to identify transcripts
rather than 11k expression
arrays based on RefSeq
gene exons
Lots of transcriptional dark matter
Non coding RNAs

ChIP-CHIP mapping of the epigenome

Protein of interest

Antibodies for acetylated or methylated histones, transcription factors,

high mobility group factors, coactivators used for mapping the distribution
of corresponding epigenetic features on the genome

Epigenomic identification of methylated DNA

Bisulfite sequencing of the genome defines actual sites of DNA methylation

but is more costly

Identification of DNAse hypersenstive sites open chromatin

Also Formaldehyde assisted isolation of regulatory elements (FAIRE) is used

isolates open instead of closed DNA

Sample of ENCODE epigenomic anotation

Replication
timing

DNAse hypersensitive
sites
Regulatory factor
Binding regions

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

Looking at ENCODE data

ES
MEF

H3K4me3

A look at multiple histone marks in the mouse genome

H3K4me3 marks at 5 end of genes

H3K36me3 marks transcriptional elongation
Aox1 expressed strongly in brain

Developmentally poised promoters in ES cells

Activating and repressive marks both present in pluripotent

Cells These represent genes with complex stage and tissue
Specific expression patterns
H3K27me3 repressive mark in differentiated cells it has the
Same pattern as H3K9Me

H3K36me3 a mark for transcriptional elongation

Differentiation

Proximal

H3K4me1
Marks insulator
DNA and
enhancers

DHS not near TSS

Transcription factor and methylated histone proximity to

transcription start sites (TSSs)

repressive mark

RFBRs regulatory factor binding regions

Evolutionary constraints in DNA sequence

Open chromatin,
Transcription factor
Binding and modified
histones

Evolutionarily conserved DNA relationship with coding sequence,

exposed chromatin and RFBRs (transcription factors and methylated histones)
contained within feature

Window surrounding feature

Protein coding

Ancient repeats
evo. neutral

Single nucleotide polymorphisms exist more frequently in regulatory

DNA sequences than in coding sequences
- more open to evolutionary change
Ancient repeats
Open chromatin

Transcription factors

SNP

Coding
sequences

Intraspecies constraint for small RNAs

Constrained sequences

This looks at differences between humans only

H3K4me2 and H3K4me3 histone marks are biased toward

Coding 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)