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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:

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slide1

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”
slide2

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
slide3

Gene silencing and why is it important

Wolffe and Matzke, Science, 1999

slide4

Epigenetics and development

n

+

n

2n DNA content

Differentiation

same DNA content,

> 200 cell types

slide5

Epigenetics and development

2n DNA content

De-differentiation?

same DNA content,

> 200 cell types

examples:

  • Cloning by nuclear transfer --> regenerate entire organism from transfer of single nucleus (e.g. Dolly)
  • 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!
slide6

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)
slide7

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

slide8

Mechanism of how DNA methylation silences gene expression:

    • steric hindrance?
    • methylated DNA recruits histone de-acetylases

TF

DNA methylation and gene silencing

slide9

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

slide10

MBD proteins interact with histone deacetylases

  • MBD2 and HDACs co-purify
  • in the same complex
  • MBD2 co-IPs with
  • HDAC activity
  • MBD2 physically co-IPs
  • with HDACs
slide12

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
slide13

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
slide14

HS-40

LCR

Gene order of globin clusters mirror expression pattern during development

slide15

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
slide16

Globin LCR and adult b-globin promoters are hyperacetylated

in adult mouse erythroid leukemia cells upon induction

Forsberg et al, PNAS, 2000

slide17

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?
slide18

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 visible

2048 identical DNA strands

slide19

X

X

Ac H4

DAPI (DNA)

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
slide20

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
slide21

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

slide22

The inactive X chromosome is depleted of K4-methylated H3,

but is enriched for K27-methylated H3

DAPI

DAPI

a-MeK27 H3

a-MeK4 H3

MeK4 H3 + DAPI

MeK27 H3 + DAPI

slide24

w+/+

w-/-

w+/+

w+/+

mosaic due to PEV

Epigenetics example #3

Position effect variegation in Drosophila

White gene encodes red pigment in eye

slide25

spreading of heterochromatin

silencing leads to inactivation of

white gene --> mosaic eye patches

Position effect variegation in Drosophila

example of epigenetic regulation since silencing of

white gene is NOT due to DNA mutation,

but due to translocation and spreading of heterochromatin

slide26

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

slide27

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

slide28

Identification of other H3 methyltransferases

  • The SET domain is the conserved catalytic core of histone methyltransferases

...

...

ARTKQTARKSTGGKAPRK

ARKSA

H3:

4

9

27

Me

Me

Me

Suv39H1/2

Su(var) 3-9

human

Drosophila

SET domain

slide29

Mutations of some histone methyltransferases cancer

Identification of H3 methyltransferases

  • The SET domain is the conserved catalytic core of histone methyltransferases

...

...

ARTKQTARKSTGGKAPRK

ARKSA

H3:

4

9

27

Me

Me

Me

MLL

Trx

Suv39H1/2

Su(var) 3-9

EZH2

E(Z)

human

Drosophila

SET domain

slide30

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

slide32

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

slide35

Ac

ARKSTGGK

...

...

H3

9

14

Histone modification-dependent recruitment of proteins

Transcriptional activation

TAFII250

Bromodomain

slide36

Histone modification-dependent recruitment of proteins

Heterochromatin assembly,

Transcriptional silencing

Transcriptional activation

HP1

TAFII250

Chromodomain

Bromodomain

Me

Ac

ARKSTGGK

...

...

H3

9

14

slide37

Histone methylation is important for

defining and maintaining epigenetic states

slide38

a = candidate approach identify by Western blotting

b = unbiased approach identify by Mass Spec

Identifying methyl-H3 binding proteins

  • histone peptide pulldown assay:

b

a

?

b

b

a

?

slide39

Me

Me

Me

...

...

ARTKQTARKSTGGKAPRK

ARKSA

H3:

4

9

27

Site specific methylation of the H3 tail has different functions

HP1

BPTF

polycomb

CD

CD

PhD

transcriptional

“competence”

transcription

repression

transcription

repression

euchromatin

constitutive

heterochromatin

facultative

heterochromatin

slide40

Heterochromatin and euchromatin

constitutive

heterochromatin

facultative

heterochromatin

euchromatin

K9-methylated H3

K27-methylated H3

K4-methylated H3

HP1

polycomb

BPTF

Yng2

slide41

HATs

Ac-histone

histone

HDACs

kinases

Phos-histone

histone

phosphatases

Different dynamics of histone modifications

highly

dynamic

HMT

more

stable

Me-histone

histone

de-methylase

slide42

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

slide43

The search for histone demethylases

  • LSD1 is a histone H3-K4 demethylase

Shi et al, Cell, 2004

slide44

The search for histone demethylases

  • LSD1 is a histone H3-K4 demethylase

Shi et al, Cell, 2004

slide45

The search for histone demethylases

Adapted from Tsukada and Zhang, Methods, 2006

slide46

Purifcation of histone demethylases

Release of radioactive

formaldehyde

Adapted from Tsukada and Zhang, Methods, 2006

slide47

Identifying site of histone demethylation

  • JHDM1A demethylates di-MeK36 on H3

Adapted from Tsukada and Zhang, Methods, 2006

slide48

Overexpression of JHDM1A results in loss of K36Me-H3

Adapted from Tsukada and Zhang, Methods, 2006

slide49

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

slide50

Epienetics and diseases

diseases

  • b-globin thalassemia
  • leukemia

adapted from Nature 429, 2004

slide51

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