heterochromatineuchromatin (and facultative heterochromatin) different types of chromatin...
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heterochromatin euchromatin(and facultative heterochromatin)
Different types of chromatin
Constitutive 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
2n DNA content
same DNA content,> 200 cell types
n n+D
iffer
entia
tion
Epigenetics and development
2n DNA content
same DNA content,> 200 cell types
De-
diffe
rent
iatio
n?
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
methyl group is added to the cytosine methylation status is maintained during replication by DNMTs
DNMTs
DNA methylation and gene silencing
Mechanism of how DNA methylation silences gene expression:
• steric hindrance?
• methylated DNA recruits histone de-acetylases
TF
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
unmethylatedprobe
methylatedprobe
shifted probes
MBD proteins interact with histone deacetylases
• MBD2 physically co-IPs with HDACs
• MBD2 co-IPs with HDAC activity
• 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 - and -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 -globin family, expression goes from - to - to -isoforms
• mutations in adult isoforms of globin genes result in thalassemia
Globin LCR and adult -globin promoters are hyperacetylated in adult mouse erythroid leukemia cells upon induction
Forsberg et al, PNAS, 2000
Epigenetics example #2Dosage 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?
bands
inter-bands
Drosophila polytene chromosomes
• Drosophila genome has 4 chromosomes
• polytene chromosomes result from endoreplication
(DNA replication without cytokinesis)
giant chromosomes that are easily visible2048
iden
tical
DN
A s
tran
ds
X chromosome in Drosophila
DAPI (DNA) Ac H4
X X
• 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 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
Jeppesen et al, Cell, 1993
metaphase chromosome immunofluorescence
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
The inactive X chromosome is depleted of K4-methylated H3,but is enriched for K27-methylated H3
MeK27 H3 + DAPIMeK4 H3 + DAPI
DAPI
-MeK4 H3
DAPI
-MeK27 H3
X inactivation involves sequential epigenetic modifications of the silenced chromosome
Epigenetics example #3Position effect variegation in Drosophila
w+/+ w-/-
w+/+ w+/+
mosaic due to PEV
White gene encodes red pigment in eye
Position effect variegation in Drosophila
spreading of heterochromatinsilencing leads to inactivation ofwhite 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
Rea et al, Nature, 2000
• The SET domain of the SUV39H1 is required for histone methyltransferase activity
and this enzyme methylates H3 at Lys9
1 9
Me Me
ARTKQTARKSTGGKAPRK ...94
H3:
Suv39H1/2Su(var) 3-9
ARKSA27
...
Me
Identification of other H3 methyltransferases
SET domain
• The SET domain is the conserved catalytic core of histone methyltransferases
human
Drosophila
Me Me
ARTKQTARKSTGGKAPRK ...94
H3:
MLLTrx
Suv39H1/2Su(var) 3-9
ARKSA27
...
Me
EZH2E(Z)
SET domain
Identification of H3 methyltransferases
• The SET domain is the conserved catalytic core of histone methyltransferases
human
Drosophila
• Mutations of some histone methyltransferases cancer
How does H3 K9-methylation functions in heterochromatin assembly?
• Su(Var) 2-5 (gene) codes for heterochromatin protein 1 (HP1)
• HP1 in Drosophila is localized to the chromocenter
HP1 DNA
• back to early genetics studies in Drosophila:
Ectopic expression of SUV39H1 causes redistribution of HP1
Melcher et al, MCB, 2000
Lys9-methylated H3 binds to the conserved motif called chromodomain
Bannister et al, Nature, 2001
• 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
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
ARKSTGGK
Ac
... ...H39 14
Bromodomain
TAFII250
Transcriptional activation
Histone modification-dependent recruitment of proteins
ARKSTGGK... ...H39 14
Me
Chromodomain
HP1
Ac
Bromodomain
TAFII250
Transcriptional activationHeterochromatin assembly,Transcriptional silencing
Histone modification-dependent recruitment of proteins
Histone methylation is important for defining and maintaining epigenetic states
Identifying methyl-H3 binding proteins
• histone peptide pulldown assay:
?
?
a
a
b
b
b
a = candidate approach identify by Western blottingb = unbiased approach identify by Mass Spec
Site specific methylation of the H3 tail has different functions
ARTKQTARKSTGGKAPRK ...94
H3: ARKSA27
...MeMe Me
CD
HP1 polycomb
CD
transcriptional “competence”
transcription repression
transcription repression
PhD
BPTF
constitutiveheterochromatin
euchromatin facultativeheterochromatin
constitutiveheterochromatin euchromatin
Heterochromatin and euchromatin
facultativeheterochromatin
K9-methylated H3 K27-methylated H3 K4-methylated H3
HP1 polycomb BPTFYng2
Different dynamics of histone Different dynamics of histone modificationsmodifications
histone Ac-histoneHATs
HDACs
histone Phos-histonekinases
phosphatases
histone Me-histoneHMT
de-methylase
highlydynamic
morestable
The search for histone demethylasesThe search for histone demethylases
luciferase
Transcription ?
5X Gal4 binding sites
• LSD1 is a transcriptional co-repressor and its repression function is mediate through the amine-oxidase domain
Shi et al, Cell, 2004
The search for histone demethylasesThe search for histone demethylases
Shi et al, Cell, 2004
• LSD1 is a histone H3-K4 demethylase
The search for histone demethylasesThe search for histone demethylases
• LSD1 is a histone H3-K4 demethylase
Shi et al, Cell, 2004
The search for histone demethylasesThe search for histone demethylases
Adapted from Tsukada and Zhang, Methods, 2006
Purifcation of histone demethylasesPurifcation of histone demethylases
Release of radioactive formaldehyde
Adapted from Tsukada and Zhang, Methods, 2006
Identifying site of histone Identifying site of histone demethylationdemethylation
Adapted from Tsukada and Zhang, Methods, 2006
• JHDM1A demethylates di-MeK36 on H3
Overexpression of JHDM1A results in loss of Overexpression of JHDM1A results in loss of K36Me-H3K36Me-H3
Adapted from Tsukada and Zhang, Methods, 2006
Histone de-methylases are found for all these sites:
LSD1JARID1a-d
JMJD2b UTXJMJD3
Apart from LSD1, all other histone de-methylases identifiedso far belong to the JmjC domain-containing family of enzymes
Epienetics and diseases
diseases
• -globin thalassemia • leukemia
adapted from Nature 429, 2004