P. Matthias, April 30, 2008
Transcription Regulation and Gene Expression in Eukaryotes FS 08
Chromatin
Bone marrow cell
DNA compaction 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• All four core histones contain a central ‘histone fold’• The histone fold is a dimerizationmotif
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)?Chromatin: DNA packing (only) or regulation (also)?
LOCI WHICH GIVE RISE TO HOMEOTIC MUTATIONS
SEGMENT SPECIFIC POLYCOMB GROUP
Suppressors of trithorax
ETP GROUP
Enhancers 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]
Comparison of genetic and epigenetic inheritance based on chromatin structuresComparison of genetic and epigenetic inheritance based on chromatin structures
‘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
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 EcoRIfragment
Chromatin-packaged DNA Chromatin-packaged DNA
Nuclease Hypersensitive Sites
The Chicken Lysozyme Gene
Dynamics of HSs
Multipotent Progenitor
Erythroblasts
Monoblasts
Promacrophages
Activated Macrophages
-2 kb-4 kb-6 kb-8 kb
[mRNA]
-
-
+
++
+++
Chromatin remodeling
Chromatin modifications
Major regulation of chromatin:
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
Chromatin remodeling complexes
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 as positive 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
HistoneHistone Modifications: Code HypothesisModifications: 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 HistonesPrevalent Epigenetic Modifications of Histones
Combinatorial histone modifications
Some specific meaning of the histone code
Some specific meaning of the histone code
How the histone code could be read by a code-reader complex
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 switchesBinary switches
Binary switchesBinary switches
Lysine methylation: not so stable after allLysine methylation: not so stable after all
Chromatin acquires additional variety through the site-specific insertion of a small set of histone variants
There are more modifications:There are more modifications:
Ubiquitination: H2B K123, monoubiquitination as signal
Arginine methylation: Protein Argine Methyltransferases(PRTM1, Carm1/PRTM4…)
ADP-ribosylation: DS-breaks,…
Modern view of the Epigenome(s)Modern view of the Epigenome(s)
ChIP-chipChIP-chipTowards a global view of chromatinTowards a global view of chromatin
ChIP-SAGEChIP-SAGETowards a global view of chromatinTowards a global view of chromatin
ChIP-SeqChIP-SeqTowards a global viewof chromatin
Towards a global viewof chromatin
Towards a global view of chromatinTowards a global view of chromatin
Towards a global view of chromatinTowards a global view of chromatin
Towards a global view of chromatin: novel “genes”Towards a global view of chromatin: novel “genes”
Modern view of the Epigenome(s)Modern view of the Epigenome(s)
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