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Different types of chromatin
heterochromatin
Constitutive heterochromatin:
euchromatin
(and facultative
heterochromatin)
• constitute ~ 10% of nuclear DNA
• highly compacted, transcriptionally inert, replicates late in S phase
Euchromatin + facultative heterochromatin:
• constitute ~ 90% of nuclear DNA
• less condensed, rich in genes, replicates early in S phase
however,
• only small fraction of euchromatin is transcriptionally active
• the rest is transcriptionally inactive/silenced (but can be activated in certain tissues or
developmental stages)
• these inactive regions are also known as “facultative heterochromatin”
Gene silencing and why is it important
• In any given cell, only a small percentage of all genes are expressed
• vast majority of the genome has to be shut down or silenced
• knowing which genes to keep on and which ones to silence is
critical for a cell to survive and proliferate normally
Gene silencing and why is it important
Wolffe and Matzke, Science, 1999
Epigenetics and development
n + n
Differentiation
2n DNA content
same DNA content,
> 200 cell types
Epigenetics and development
De-differentiation?
2n DNA content
same DNA content,
> 200 cell types
examples:
1. Cloning by nuclear transfer --> regenerate entire organism from
transfer of single nucleus (e.g. Dolly)
2. Induced pluripotent stem cells (iPS) --> expression of 4 genes are
sufficient to transform differentiated cells to “stem” cells

Both processes must involve reprogramming of epigenome!
Epigenetics and epigenetic regulation
Definition of Epigenetics:
• heritable changes in gene expression that do not involve changes
in DNA sequences
• mechanisms:
• DNA methylation
• histone modifications
• examples:
• Developmentally regulated or tissue specific gene expression
• X chromosome dosage compensation
• Drosophila position effect variegation (PEV)
Epigenetic mechanism #1: DNA methylation
• DNA methylation has long been correlated with repression of gene expression
• DNA methylation mostly occurs on CpG dinucleotides
DNMTs
methyl group is added to the cytosine
methylation status is maintained
during replication by DNMTs
DNA methylation and gene silencing
Mechanism of how DNA methylation silences gene expression:
• steric hindrance?
TF
• methylated DNA recruits histone de-acetylases
A class of proteins called MBD bind methylated DNA
• MeCP2 is the first protein found to bind to methylated DNA
• mutation of MeCP2 gene causes Rett Syndrome in humans
shifted
probes
unmethylated
probe
methylated
probe
MBD proteins interact with histone deacetylases
• MBD2 co-IPs with
HDAC activity
• MBD2 physically co-IPs
with HDACs
• MBD2 and HDACs co-purify
in the same complex
DNA methylation recruits histone deacetylases
Epigenetic mechanism #2: histone methylation
• histone H3 is methylated at several lysine residues
• H3 K4-methylation is associated with transcriptional activation
whereas K9-, K27-methylation is associated with repression
• these H3 methylation sites define the transcriptional/epigenetic states
of the associated genes/chromatin domains
Epigenetics example #1:
Tissue-specific and developmentally regulated gene
expression
• globin genes are expressed only in erythroid cells
• hemoglobin made up of 2 copies each of a- and b-chains
Gene order of globin clusters mirror expression pattern during development
HS-40
LCR
Globin genes are tissue-specific and developmentally regulated
• Distinct isoforms of the globin genes are expressed at different developmental stages
• e.g., for the b-globin family, expression goes from e- to g- to b-isoforms
• mutations in adult isoforms of globin genes result in thalassemia
Globin LCR and adult b-globin promoters are hyperacetylated
in adult mouse erythroid leukemia cells upon induction
Forsberg et al, PNAS, 2000
Epigenetics example #2
Dosage compensation of X chromosome
• for many organisms, females have 2 copies of the X chromosome whereas males
only have single copy
• how to balance expression dosage of X-linked genes?
2048 identical DNA strands
Drosophila polytene chromosomes
bands
• Drosophila genome has 4 chromosomes
• polytene chromosomes result from endoreplication
(DNA replication without cytokinesis)
inter-bands
 giant chromosomes that are easily visible
X chromosome in Drosophila
• the X chromosome of male Drosophila is transcriptionally twice as active
• increased transcription of the active X chromosome is marked by
hyper-acetylated histones
X
X
DAPI (DNA)
Ac H4
X chromosome inactivation
• In female mammals, one of the two X chromosomes in the genome is transcriptionally inactivated
in order to equalize expression of X-linked genes in males and females (dosage compensation)
• Inactivation of the maternal or paternal chromosome is random
X chromosome inactivation
• In X inactivation, almost the entire X chromosome is transcriptionally silenced
• Transcriptional silencing of this chromosome correlates with distinct histone modification patterns
• eg. histone H4 is hypo-acetylated on the inactive X chromosome
metaphase chromosome immunofluorescence
Jeppesen et al, Cell, 1993
The inactive X chromosome is depleted of K4-methylated H3,
but is enriched for K27-methylated H3
DAPI
DAPI
a-MeK4 H3
a-MeK27 H3
MeK4 H3 + DAPI
MeK27 H3 + DAPI
X inactivation involves sequential epigenetic modifications of the
silenced chromosome
Epigenetics example #3
Position effect variegation in Drosophila
White gene encodes red pigment in eye
w+/+
w-/-
w+/+
w+/+
mosaic due to PEV
Position effect variegation in Drosophila
spreading of heterochromatin
silencing leads to inactivation of
white gene --> mosaic eye patches
example of epigenetic regulation since silencing of
white gene is NOT due to DNA mutation,
but due to translocation and spreading of heterochromatin
Position effect variegation in Drosophila
Su(var) mutations = Suppressors of PEV
e.g. Su(var)2-5 = HP1
Su(var)3-9 = SET-domain protein
Identification of H3 Lys9 methyltransferase
• The first lysine-specific HMT was identified by IP-in vitro activity assays
1
9
• The SET domain of the SUV39H1 is required for histone methyltransferase activity
and this enzyme methylates H3 at Lys9
Rea et al, Nature, 2000
Identification of other H3 methyltransferases
• The SET domain is the conserved catalytic core of histone methyltransferases
H3:
... ARKSA ...
ARTKQTARKSTGGKAPRK
4
9
27
Me
Me
Me
Suv39H1/2
Su(var) 3-9
SET domain
human
Drosophila
Identification of H3 methyltransferases
• The SET domain is the conserved catalytic core of histone methyltransferases
H3:
... ARKSA ...
ARTKQTARKSTGGKAPRK
4
9
27
Me
MLL
Trx
Me
Me
Suv39H1/2
Su(var) 3-9
EZH2
E(Z)
SET domain
• Mutations of some histone methyltransferases
cancer
human
Drosophila
How does H3 K9-methylation functions in heterochromatin assembly?
• back to early genetics studies in Drosophila:
• Su(Var) 2-5 (gene) codes for heterochromatin protein 1 (HP1)
• HP1 in Drosophila is localized to the chromocenter
HP1
DNA
Ectopic expression of SUV39H1 causes redistribution of HP1
Melcher et al, MCB, 2000
Lys9-methylated H3 binds to the conserved motif called chromodomain
• Using the peptide pull-down assay, it was found that Lys9-methylated H3 binds to
heterochromatin protein 1 (HP1)
• HP1 is a protein previously identified to be enriched in and important for
heterochromatin assembly
• Lys9-methylated H3 binds to HP1 via the chromodomain motif in HP1
Bannister et al, Nature, 2001
H3 K9-methylation is required for HP1 localization
Lachner et al, Nature, 2001
H3 K9-methylation is required for HP1 localization
Lachner et al, Nature, 2001
Histone modification-dependent recruitment of proteins
Transcriptional activation
TAFII250
Bromodomain
Ac
H3
... ARKSTGGK ...
9
14
Histone modification-dependent recruitment of proteins
Heterochromatin assembly,
Transcriptional silencing
Transcriptional activation
HP1
TAFII250
Chromodomain
Bromodomain
Me
H3
Ac
... ARKSTGGK ...
9
14
Histone methylation is important for
defining and maintaining epigenetic states
Identifying methyl-H3 binding proteins
• histone peptide pulldown assay:
b
a
?
b
b
a
?
a = candidate approach
b = unbiased approach
identify by Western blotting
identify by Mass Spec
Site specific methylation of the H3 tail has different functions
BPTF
PhD
Me
H3:
HP1
polycomb
CD
CD
Me
Me
... ARKSA ...
ARTKQTARKSTGGKAPRK
4
9
27
transcriptional
“competence”
transcription
repression
transcription
repression
euchromatin
constitutive
heterochromatin
facultative
heterochromatin
Heterochromatin and euchromatin
constitutive
heterochromatin
K9-methylated H3
HP1
facultative
heterochromatin
K27-methylated H3
polycomb
euchromatin
K4-methylated H3
BPTF
Yng2
Different dynamics of histone modifications
HATs
Ac-histone
histone
HDACs
highly
dynamic
kinases
Phos-histone
histone
phosphatases
HMT
Me-histone
histone
de-methylase
more
stable
The search for histone demethylases
• LSD1 is a transcriptional co-repressor and its repression function is
mediate through the amine-oxidase domain
Transcription
?
luciferase
5X Gal4 binding sites
Shi et al, Cell, 2004
The search for histone demethylases
• LSD1 is a histone H3-K4 demethylase
Shi et al, Cell, 2004
The search for histone demethylases
• LSD1 is a histone H3-K4 demethylase
Shi et al, Cell, 2004
The search for histone demethylases
Adapted from Tsukada and Zhang, Methods, 2006
Purifcation of histone demethylases
Release of radioactive
formaldehyde
Adapted from Tsukada and Zhang, Methods, 2006
Identifying site of histone demethylation
• JHDM1A demethylates di-MeK36 on H3
Adapted from Tsukada and Zhang, Methods, 2006
Overexpression of JHDM1A results in loss of K36MeH3
Adapted from Tsukada and Zhang, Methods, 2006
Histone de-methylases are found for all these sites:
LSD1
JARID1a-d
JMJD2b
UTX
JMJD3
Apart from LSD1, all other histone de-methylases identified
so far belong to the JmjC domain-containing family of enzymes
Epienetics and diseases
diseases
• b-globin thalassemia
• leukemia
adapted from Nature 429, 2004
Paper assignments for Nov 2nd:
Group 1 (Chanda - Fasih):
Kuzmichev et al, 2002, Genes Dev. 16: 2893-2905
Histone methyltransferase activity associated with a human multiprotein
complex containing the enhancer of Zeste protein
Group 2 (Fenton - How):
de Napoles et al, 2004, Dev. Cell 7: 663-676
Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to
heritable gene silencing and X inactivation
Group 3 (Karisch - Yan):
Wysocka et al, 2006, Nature 442: 86-90
A PHD finger of NURF couples histone H3 lysine 4 trimethylation with
chromatin remodeling