Transcription Regulation And Gene Expression in Eukaryotes

Cycle G2 (lecture 13709)

P. Matthias, April 28th, 2010

Chromatin

www.fmi.ch/training/teaching

Chromatin-nucleosomesChromatin: packaging vs regulationNucleosome remodelingHistone modifications / codeHistone variantsCurrent view

Bone marrow cell

DNA compaction in a human nucleus

1bp (0.3nm)

10,000 nm

30nm

11 nm

•A human nucleus is ~5-8 µm in diameter and contains ~ 2 meters of DNA

Nucleosomes

• Most of the interphase chromatin exists as a 30 nm fiber. If this chromatin is unfolded it appears as “beads on a string.”

• The string is the DNA and each bead corresponds to a nucleosome core particle.

• An individual nucleosome core particle consists of a complex of eight histone proteins (2x H2A, H2B, H3, & H4) and ~ 146 bp.

• This shortens the DNA about 1/3 and is the first level of packing.

30 nm fiber

Genes VIII Chapter 20

Genes VIII Chapter 20

Genes VIII Chapter 20

Histone Structure: histone fold

• All four core histones contain a central ‘histone fold’• The histone fold is a dimerization motif

Histone octamer assembly

H3-H4tetramer H2A-H2B

dimer

Histoneoctamer

Nucleosome Structure (X-ray)

Consequences of DNA Packaging

• Distortions make it often impossible for gene-specific transcription factors to bind to their target sequences

• Not all DNA sequences can be sufficiently distorted for packaging into nucleosomes– nucleosome-free regions

Chromatin: DNA packing (only) or regulation (also)?

LOCI WHICH GIVE RISE TO HOMEOTIC MUTATIONS

SEGMENT SPECIFIC POLYCOMB GROUP

Suppressors of trithorax

ETP GROUP

Enhance rs of trithoraxand Polycomb

TRITHORAX GROUP

Suppressors of Polycomb

Ultrabithorax(Ubx)

Polycomb(Pc)

Enhancer of zeste[E(z)]

trithorax(trx)

Abdominal-A(Abd-A)

polyhomeotic(ph)

Additional sex combs(Asx) (ash1)

Abdominal-B(Abd-B)

Polycomb-like(Pcl) ? (ash2)

pleiohomeotic(pho)

Enhancer of Polycomb[E(Pc)]

brahma(brm)

Antennapedia(Antp)

multi sex combs

(mxc)

Sex combs on midleg(Scm

moira(mor)

Sex combs reduced(Scr)

extra sex combs(esc)

Sex combs extra(Sce)

kismet(kis)

proboscipedia(pb)

super sex comb s(sxc)

Posterior sex combs (Psc)(mod mdg4)

Deformed(Dfd)

Suppressor of zeste-2[su(z)2] (lawc)

Labial(Lab)

Suppressor of zeste-4[su(z)3]

Suppressor of zeste-6[su(z)4]

Suppressor of zeste-7[su(z)7]

‘Indirect Endlabeling’ Method

EcoRI EcoRIDNAase I

Hybridization Probe

1. Cut HS with DNAase I in purified nuclei

2. Purify DNA and recut with a restriction enzyme: EcoRI

3. Carry out a Southern blot with a probeadjacent to restriction enzyme target site

Minus DNAase I

Plus DNAase I

Size of fragment reveals location of HS relative to probed terminus

Full-length EcoRI fragment

Chromatin-packaged DNA Chromatin-packaged DNA

Nuclease Hypersensitive Sites

The Chicken Lysozyme Gene

Dynamics of DNAseI Hypersensitive Sites

Multipotent Progenitor

Erythroblasts

Monoblasts

Pro-macrophages

Activated Macrophages

-2 kb-4 kb-6 kb-8 kb

[mRNA]

-

-

+

++

+++

Chromatin remodeling

Chromatin modifications

Major regulation of chromatin:

Nucleosomes are dynamic

Chromatin Remodelling

• Replication-dependent– as DNA gets replicated, nucleosomes dissociate from the DNA to

leave regulatory regions free of nucleosomes -> brief ‘window of opportunity’ to establish new chromatin configurations

• Replication-independent– allows genes to be switched on and off regardless of cell cycle

state

SWI/SNF and others: Chromatin Remodeling Complexes

• The SWI/SNF chromatin remodeling activity, together with the related RSC, NURD, NURF, Mi-2, and CHRAC complexes, utilizes ATP to alter nucleosomal structure– Each member of the ATP-dependent family of chromatin

remodeling enzymes contains an ATPase subunit that is related to the SWI2/SNF2 subfamily of the DEAD/H box superfamily of nucleic acid-stimulated ATPases

Effects on Gene Expression

• Most SWI/SNF-like enzymes play direct roles in the activation of transcription– In yeast, genes that encode many of the eleven subunits of the

ySWI/SNF complex were initially identified in genetic screens aspositive regulators of transcription

• Genome-wide expression analyses have shown that inactivation of ySWI/SNF leads to decreases in steady-state transcription of around 5% of the approximately 6000 yeast genes

Chromatin remodeling complexes

Chromatin remodeling complexes

Chromatin Remodelling

Histone Modifications: Code Hypothesis

• As proposed by Allis and Strahl: “that multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions”

Strahl, B.D. and Allis, C.D., Nature. 2000

Different modifications

Prevalent Epigenetic Modifications of Histones

Combinatorial histone modifications

Some specific meaning of the histone code

How the histone code could be read by a code-reader complex

Zhang & Reinberg (2001) Genes & Dev. 2343-2360.

Model for H3K9 Methylation in silencing of heterochromatin and euchromatin.

Binary switches

Binary switches

Lysine methylation: not so stable after all

Additional chromatin variety: histone variants

Modern view of the Epigenome(s)

ChIP-chipTowards a global view of chromatin

ChIP-SAGETowards a global view of chromatin

ChIP-SeqTowards a global viewof chromatin

Towards a global view of chromatin

Towards a global view of chromatin

Towards a global view of chromatin: novel “genes”

Modern view of the Epigenome(s)

Comparison of genetic and epigenetic inheritance based on chromatin structures

There are more modifications:

Ubiquitination: H2B K123, monoubiquitination as signal

Arginine methylation: Protein Argine Methyltransferases (PRTM1, Carm1/PRTM4…)

ADP-ribosylation: DS-breaks,…