cycle g2 (lecture 13709) p. matthias, april 28th, 2010
TRANSCRIPT
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,…
