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