Nucleosome structure

Chromatin folding

polytenic chromosomes

32x polyteny

256x polyteny

Tip of the X

Genome Browsers

The Drosophila X chromosome displayed on the Affymetrix Integrated Genome Browser

telomere centromere

z w

z

w

The zeste-white region

3A 3C

w

w

w

Heat shock puffs

Puffs accumulateRNA polymerase

Puffs are sites of active transcription

Interbands are like small puffs

RNA polymerase

epigenomes

Nucleosome structure

epigenetic memory

Partial (variegated) silencing of the white genein presence of a PRE

Other examples of epigenetic information

HeterochromatinCentromere functionMammalian X inactivationImprinted genes

Chromatin modifications

acetylationphosphorylationmethylationubiquitylation

Position and integrity of nucleosomes

Modifications of histones

Histone variants

DNA methylation

Binding of chromatin proteins

Histone tails

Lysine methylation

HMTs

Transcription marks

Thechromodomain

Chromodomains are structuralmotifs that form a pocket lined withhydrophobic residues into which themethylated lysine fits.They are therefore methyllysinebinding domains. Heterochromatinprotein HP1 and Polycomb proteinPC are two examples ofchromodomain-containing proteins

Chromatin immunoprecipitation

Crosslink chromatin

Fragment chromatin

Add antibody Ab

Collect all Ab binding fragments

Reverse crosslinking and purify DNA

Detect DNA sequences

H3 K4 methylation

H3 K4 methylation

dRING

PC

PH

PSC

includes a core quartet of proteins:

Polycomb PC

Posterior sex combs PSC

Polyhomeotic PH

dRING

chromodomain binds H3 meK27

RING domain ubiquitylates H2A

PRC1: PC complexPRC1: PC complex

E(Z)

p55RPD3

ESC

Su(z)12

Enhancer of zeste E(Z) SET domain H3 methyltransferase

includes :

E(Z)

PRC2: E(Z) complexPRC2: E(Z) complex

The E(z)/Esc complex possessesThe E(z)/Esc complex possesseshistone histone H3 H3 methyltransferase methyltransferase activityactivity

ARTARTKKQTARQTARKKSTGGKAPRKQLATKAARSTGGKAPRKQLATKAARKKSAPATGGVK-SAPATGGVK-44 272799

H3

E(z)/Esc complexE(z)/Esc complex (~600kDa)(~600kDa)

p55

ESCE(z)

RPD3Su(z)12

CHCH33 CHCH33CHCH33

It can mono-, di- or trimethylate lysine 27

The E(z)/Esc complex possessesThe E(z)/Esc complex possesseshistone histone H3 H3 methyltransferase methyltransferase activityactivity

H3 K27me1H3 K27me2H3 K27me3

The three degrees of methylation mean different things

H3 K27me1 and H3 K27me2 are almost everywhereIn the genome, as can be seen in polytene chromosomesstained with antibody against H3 K27me2

H3 K27 dimethylation

α-H3 me2K27

Ebert et al. 2004

H3 K27 dimethylation

The E(z)/Esc complex possessesThe E(z)/Esc complex possesseshistone histone H3 H3 methyltransferase methyltransferase activityactivity

H3 K27me3 is found only at certain specificsites

These turn out to be the sites where Polycombcomplexes bind

α-H3 me3K27

Ebert et al. 2004

H3 K27 trimethylation

How do PcG complexes silence?

PRE promoter

PRE

How do PcG complexes silence?

What recognizes the What recognizes the histonehistonemethylation methylation mark?mark?

The PC chromodomain has been shown to bind toH3 methylated at lysine 27

The affinity to K27me3 is 5x that to K27me2This could contribute to target and stabilize PC binding

me3K27 H3me3K27 H3me3K27 H3

ModelModel

PH

PSC PCdRING

ESC

E(z)

ModelModel

PH

PSC PCdRING

ESC

E(z)

ModelModel

PH

PSC

dRING

ESC

E(z)

PC

ModelModel

PHPSC

dRING

ESC

E(z)

PC

ModelModel

PH

PSC PCdRING

ESC

E(z)

PH

PSC

PC

dRIN

G

ModelModel

dco-Sox100 region

me3K27

PC

PSC

E(Z)

dco-Sox100 methylation

me3K27

me3K4

Pol II

TRX

Selected Selected PcG PcG target genestarget genes

engrailed transcription factor pattern formationwingless morphogen pattern formationdpp TGF-β growth factor pattern formationhedgehog morphogen pattern formationforkhead transcription factor cell fate specificationeyeless transcription factor cell fate specificationtinman transcription factor heart formationKrüppel transcription factor cell fate specificationOr85d receptor olfactory receptor

polyhomeotic PcG gene chromatin silencingPsc PcG gene chromatin silencingpolyhomeotic PcG gene chromatin silencingPsc PcG gene chromatin silencing