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Chromatin
Chromosomes and
Chromosomes
Chromatin
- Structure
composed of a very long DNA molecule and
associated proteins that carries the genetic (and epigenetic)
information
- Especially evident in plant and animal cells undergoing mitosis
or meiosis, where each chromosome becomes condensed into a
compact rod-like structure that is visible by light microscopy
Chromatin
- Complex of DNA, histones and non-histone proteins that
collectively make up chromosomes
- DNA, histones and non-histone proteins are subject to posttranslational/replication modifications that form the basis of an
epigenetic code
B. A replicated chromosome
at mitosis
levels of
Chromatin compaction
Most interphase
chromatin is condensed
into 30nm coils.
Interphase chromatin
~500 fold compaction
end-to-end
Mitotic chromatin 20X fold
compaction end-to-end
over interphase
A) 30 nm fibers
B) beads on a string-nucleosome
from interphase nucleus
Nomenclature
Nucleosome = a nucleosome core particle + linker
DNA+ a linker histone
DNA length: 180-200 bp
Nucleosome core particle = histone octamer + 146 bp DNA
H1
Molecular
weight
21
Major basic
Amino acids
++
Lys
H2a
13.8
Lys
H2b
13.8
Lys
H3
15.4
Arg/Lys
H4
11.4
Arg/Lys
Nucleosome Positioning
linker
Core particle
Translational positioning
Rotational Positioning
Article
Nature 442, 772-778 (17 August 2006)
Abstract
Eukaryotic genomes are packaged into nucleosome particles that occlude the
DNA from interacting with most DNA binding proteins. Nucleosomes have higher
affinity for particular DNA sequences, reflecting the ability of the sequence to bend
sharply, as required by the nucleosome structure. However, it is not known
whether these sequence preferences have a significant influence on
nucleosome position in vivo, and thus regulate the access of other proteins
to DNA. Here we isolated nucleosome-bound sequences at high resolution from
yeast and used these sequences in a new computational approach to construct
and validate experimentally a nucleosomeDNA interaction model, and to predict
the genome-wide organization of nucleosomes. Our results demonstrate that
genomes encode an intrinsic nucleosome organization and that this
intrinsic organization can explain approx. 50% of the in vivo nucleosome
positions. This nucleosome positioning code may facilitate specific
chromosome functions including transcription factor binding, transcription
initiation, and even remodelling of the nucleosomes themselves.
Naked DNA
Level of accessibility/
Transcriptional activity
High
low
Unavoidably
High levels of
Transcription
Mechanisms for
decreased
accessibility or
repression
Mechanisms for
increased
accessibility or
activation
Chromatin
Remodeling by ATP-driven
chromatin remodeling complexes
Yeast SWI/SNF
10 proteins
Needed for expression of genes involved in mating-type
switching and sucrose metabolism (sucrose non-fermenting).
Some suppressors of swi or snf mutants are mutations in
genes encoding histones.
Interacts with chromatin to activate a subset of yeast genes.
Is an ATPase-containing complex
Involvement of SWI/SNF in
transcription
Structural alteration
Nucleosome sliding
Nucleosome eviction
The consequence of chromatin remodeling is
dependent on the type of chromatin
remodeling complexes involved
NURF +ATP
TF
NURF +ATP
Evenly space
nucleosomes that
would otherwise
clump due to
influence of DNA
sequence
Chromatin remodeling by
covalent modification of
histones
coA
Histone Methylation
Two chemical classes of HMTs: arginine specific-HMTs
and lysine-specific HMTs.
Both Arg and Lys can be mono-, di- or tri-methylated.
In contrast to histone acetylation, histone methylation is
believed to be stable until recently. This modification is
still rather stable though. Turnover rate of histone
methylation is similar to that of histone turnover
Methylation does not neutralize positive charge on Lys
and Arg.
Methyl group is thought to be too small to have a direct
effect on chromatin structure.
The function of histone methylation is believed to be
mediated through specific methylated histone binding
proteins (as part of histone code hypothesis).
SummaryofKnownHMTasesandTheirTargetSites
HMTase
Histones
Sites
R2, R17, R26
Roles in transcription
CARM1
H3
activation
PRMT1
H4
R3
SUV39H1/Clr4/ESET H3
K9
silencing/X-inactivation
ySET1/mSET7
H3
K4
ySET2
H3
K36
elongation
G9a
H3
K9
silencing
EZH1/EZH2
H3
K27
silencing/X-inactivation
H3
K79
activation
SET8
H4
K20
Specific demethylation
Histone phosphorylation
Phosphorylation on Ser-10 of H3 is involved in
both transcriptional activation and in chromosome
condensation during mitosis
Phosphorylation on Ser-10 of H3 may facilitate
acetylation of histone H3 on Lys-9 and Lys-14
Phosphorylation of histone H2AX variant is
involved in DNA repair
Phosphorylation of Ser14 of H2B is linked to
apoptosis
Many Ser and Thr sites in histone tails can be
phosphorylated. In most cases the functional
consequence is not clear
Histone Ubiquitination
H2A (Lys-119) and H2B (Lys-123) can be monoubiquitinated
Mono-ubiquitination is not associated with protein
degradation by the proteasome
H2B ubiquitination in yeast is catalyzed by Rad6
and is required for methylation on Lys-4 and Lys79 of H3 (trans-histone effect). The underlining
mechanism is unknown.
Both ubiquitination and de-ubiquitination are
involved in transcriptional regulation (Genes and
Development 17: 2648-2663, 2003)
InterplayBetweenDifferentHistoneModifications
Human H3
Me Me
Me
Ac p
Ac
MeAc
Ac
Me Me p
14
18
23
27
NARTKQTARKSTGGKAPRKQLATKAARKSAP...
4
Me
2nd
Human H4
Me Ac
Ac
Ac
Ac
Me
3 5
12
16
20
AcNSGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...
PRMT1
1st
p300
TheHistoneCode
Histone code hypothesis: that multiple histone modifications,
acting in combinatorial or sequential fashion on one or multiple
histone tails, specify unique downstream functions.
How the histone code be read?
Likely read by specific protein domains
Code
Protein Motif
Ac-Lys
H3K9Me
HP1 Chromo
H3K27Me
Polycomb Chromo
H3K4Me
Phos-H3 S10
Different combinations
22.228 lecutre 7
47
Epigenetics
Epigenetics is the study of heritable mechanisms
that affect the transcriptional state of a gene which
is not due to change in DNA sequence
Molecular mechanisms that mediate epigenetic
phenomena include (but is not limited to): DNA
methylation, histone modifications and RNAi
machinery (specific chromatin structures that allow
stable transcriptional activation or silencing)
Figure 9-65. Position-effect variegation in Drosophila. (A) Heterochromatin (red) is normally prevented from spreading into adjacent
regions of euchromatin (green) by special barrier sequences of unknown nature. In flies that inherit certain chromosomal translocations,
however, this barrier is no longer present. (B) During the early development of such flies, the heterochromatin now spreads into neighboring
chromosomal DNA, proceeding for different distances in different cells. The spreading soon stops, but the established pattern of
heterochromatin is inherited, so that large clones of progeny cells are produced that have the same genes condensed into heterochromatin and
thereby inactivated (hence the "variegated" appearance of some of these flies; see Figure 9-51B). This phenomenon shares many features with
X-chromosome inactivation in mammals.
Insulator
methylation
Insulator/boundary
element
Living cell
FormalinCross linked
chromatin
Ligate, then
Reverse cross
links and sequence
ChIA-PET clones
anchor gene
Loop gene
anchor gene
Dosage Compensation
Males (XY) and females (XX) must generate equal
amounts of most X-linked gene products
Three distinct mechanisms:
Mammals: one of X chromosome is inactivated
D. melanogaster: Males double transcription from the
single X chromosome to equal XX females
C. elegans: Females reduce transcription from both XX by
half to equal XO males
1.7 kb ncRNA
In mammals, Xist RNA is transcribed from the X inactivation center
(XIC) and spreads to inactivate the X chromosome in cis. Xist transgenes
inserted on autosomes also cause the spreading of transcriptional
inactivation in cis.
In flies, there are about 35 chromatin entry sites (CES) on the X
chromosome, including roX1 and roX2. Unlike the XIC in mammals, the
entry sites in flies regulate twofold transcriptional activation.
Epigenomics -
Epigenetics -
Omics -
Genomics
- Primary DNA sequence
- Evolutionary conservation of primary sequence
- Shared synteny - positional co-localization of genes
on chromosomes of different species i.e., Hox cluster
- Copy number variations, indels
- SNPs single nucleotide polymorphisms
Phenotype
Transcriptome transcriptional
output mRNA, miRNA, ncRNA
Transcriptome profiling
Transcriptome profiling:
224K or tiling arrays used
to identify transcripts
rather than 11k expression
arrays based on RefSeq
gene exons
Lots of transcriptional dark matter
Non coding RNAs
Protein of interest
Replication
timing
DNAse hypersensitive
sites
Regulatory factor
Binding regions
http://genome.ucsc.edu/ENCODE/encode.hg18.html
ES
MEF
H3K4me3
Differentiation
Proximal
H3K4me1
Marks insulator
DNA and
enhancers
repressive mark
Open chromatin,
Transcription factor
Binding and modified
histones
Protein coding
Ancient repeats
evo. neutral
Transcription factors
SNP
Coding
sequences
Constrained sequences
100
X
# of tissues and cell lines (NCI 60, others from ATCC, ES cells)
100
X
# of biological conditions to assess (G1/S/G2/M, hormone treatment, growth factors)
$5000
= 100 x 100 x 100 x $5000 = $5,000,000,000 ($5 billion)
The End