Download Chromatin Structure 1

Chromatin structure
Genome organisation at the DNA level
Genome is plastic
Genes may be available for expression in some cells but not in
the others (or at some time in the development but not others)
Genes may be amplified or made more available than usual
under some conditions
Change in physical arrangement of DNA (levels of DNA
packing) affect gene expression – genes in heterochromatin
and mitotic chromosomes are not expressed.
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Genome organisation at the DNA level
DNA in eukaryotic genomes is organised differently from that in
prokaryotes
In prokaryotes, most DNA codes for protein (mRNA), tRNA or
rRNA, and coding sequences are not interrupted.
In eukaryotes, most DNA does not encode protein or RNA, and
coding sequences may be interrupted by noncoding DNA
(introns).
Chromatin structure
Chromatin Structure is based on successive level of DNA packing.
Prokaryotic DNA is:
Usually circular
Much smaller – small nucleoid region
Associated with only a few protein molecules
Less elaborately structured and folded: DNA-protein loops
anchored to the plasma membrane
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Chromatin structure
Chromatin Structure is based on successive level of DNA packing.
Eukaryotic DNA is:
Complexed with a large amount of protein to form
chromatin
Highly extended and tangled during interphase
Condensed into short, thick, discrete chromosomes during
mitosis
Enormous amount of DNA requires an elaborate system of
DNA packing to fit all of the cell’s DNA into the nucleus
Genome packaging: Chromosomes - DNA and associated protein,
which together are called chromatin.
chromatin
Two types of proteins in chromatin: histones and nonhistone
proteins.
Nonhistone proteins: diverse structural, enzymatic, and regulatory
proteins.
Histones: Packaging of eukaryotic DNA depends on histones.
Approx. 10% of the chromatin remains in a condensed, compacted
form throughout interphase. This compacted chromatin is seen at the
periphery of the nucleus.
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Chromatin Structure and Transcription
Transcriptionally active chromatin has distinctive properties compared
to inactive chromatin.
Active chromatin is more accessible to enzyme degradation (DNAse
I), at hypersensitive sites. These sites are found typically in the
regulatory regions of actively transcribed, but they are absent from the
same regions of genes that are transcriptionally silent.
Chromatin Structure
Composition: DNA + associated proteins
1. Histones (small, basic; more details below)
2. Nonhistone regulatory proteins
States of chromatin
1. In all states have proteins attached
2. Usually differences are due to different states of folding after
histones added, not removal of histones
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Two Basic States of Chromatin
Heterochromatin
1. Darkly stained, relatively condensed, genetically inactive
2. Two kinds of (interphase) heterochromatin
•Constitutive heterochromatin -- always heterochromatic (ex:
chromatin at centromeres, telomeres). Usually repeating in
sequence, non coding.
•Facultative heterochromatin- sometimes heterochromatic
(depends on tissue, time etc.). Example: inactive X.
3. All DNA (chromatin) is heterochromatic during mitosis.
•Constitutive heterochromatin remains in the compacted state
in all cells at all times (DNA that is permanently silenced). The
bulk of the constitutive heterochomatin is found in and around
the centromere of each chromosome in mammals. The DNA of
constitutive heterochromatin consists primarily of highly
repeated sequences and contains relatively few genes. When
genes that are normally active are transposed into a position
adjacent to heterochromatin, they tend to become inactivated.
•Facultative heterochromatin is chromatin that has been
specifically inactivated during certain phases of an organism’s
life. Although cells of females contain two X chromosomes, only
one of them is transcriptionally active. The other X chromosome
remains condensed as a heterochromatic clump called a Barr
body.
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Euchromatin
1. Stains more lightly, less condensed.
2. Capable of genetic activity (transcription). Normal state of
most DNA during interphase.
3. Euchromatin is often divided into several distinguishable
states of folding (although tightness of folding is probably really
continuous from relatively loose to relatively tight). Correlation
between folding and function is complex.
Histones
•5 types:
9H2A, H2B (slightly lys rich),
9H3,
9H4 (arg rich)
9H1 (lys rich). All relatively small proteins.
•Per 200 bp of DNA: 2 molecules each of H2A, H2B, H3, H4 and
one molecule of H1.
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Model for Chromatin - Beads on string level
1. Octamer of 2 each of H2A, H2B, H3, H4 (+ some DNA) one bead.
2. DNA wound 2X around (on outside of) each octamer.
3. Linker DNA between cores - 50-60 bp; most exposed and
most sensitive to Dnase enzyme
4. H1 is on outside of DNA/octamer
5. Nucleosome - repeating unit - 200 BP DNA + octamer (H1
opt).
Nucleosomes & Higher Levels of Structure (requires H1).
1. Nucleosomes. DNA + histones - Chain of nucleosomes (1/7)
2. Solenoid. Chain of nucleosomes - 30 nm fiber (supercoil or
solenoid 3 beads across; 6 beads/turn) - (1/42). Need tails
(of histones) and H1 to form 30nm fiber.
3. Loops. 30 nm fiber - loops about 300nm in diameter (1/750
orig. length). Different sections may be tighter or looser.
NB: Individual loops are stretched out (probably to beads-on-astring stage) when actually transcribed.
4. Higher Orders of Folding. Looped structure folds further heterochromatin (not transcribable)
• Form structures/fibers about 700 nm across (per chromatid)
• At metaphase = tightest = 1/15,000-1/20,000 orig. length
Chromosome is 4-5 microns long but contains 75 mm of
double helical DNA.
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Levels of chromatin
packing
(a) DNA and histone
molecules form "beads
on a string," consisting
of nucleosomes in an
extended configuration.
Each nucleosome has
two molecules each of
four types of histone,
around which the DNA
wraps.
The fifth histone, called
H1, can bind to DNA
adjacent to a "bead."
Levels of chromatin
packing
(b) With the help of
histone H1, the string
of nucleosomes coils to
form a chromatin fiber
that is 30 nm in
diameter.
(c) At the next level are
looped domains of the
30-nm fiber.
The loops are attached
to a scaffold of
nonhistone proteins.
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Levels of
chromatin packing
(d) The chromatin
folds further, resulting
in the maximally
compacted
chromosome seen at
metaphase.
Nucleosomes
Proteins called histones are responsible for the first level of DNA
packing in eukaryotic chromatin.
Histones have high proportion of positively charged amino acids
(lysine and arginine), and they bind tightly to the negatively
charged DNA.
The basic unit of DNA packing is a nucleosome.
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Nucleosomes
Nucleosomes in electron micrographs look like beads on a string.
The nuclosome bead consists of DNA wound around a protein
core composed of two molecules each of four different types of
histone: H2A, H2B, H3 and H4.
The fifth histone, called H1, attaches to the DNA near the bead
when the chromatin undergoes the next level of packing.
Ribbon diagram of the nucleosome
H2A – yellow; H2B – red; H3 – blue, H4 - green
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Space filling model of the nucleosome
H2A – yellow; H2B – red; H3 – blue, H4 - green
Solenoid model of the 30nm condensed chromatin fiber in a side view.
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Histone Acetylation
Histone Acetylation is a way the nucleosome may be restructured by
addition of acetyl groups to the lysine residues of the core histones.
Acetylated lysine side chains no longer bear a positive charge and lose
their affinity to DNA
Acetylation of the lys at the N terminus of histone proteins removes
positive charges, thereby reducing the affinity between histones and
DNA. This makes RNA polymerase and transcription factors easier to
access the promoter region. Therefore, in most cases, histone acetylation
enhances transcription while histone deacetylation represses transcription.
Histone acetylation is catalyzed
by histone acetyltransferases
(HATs) and histone
deacetylation is catalyzed by
histone deacetylases (denoted
by HDs or HDACs)
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DNA methylation and gene repression
One out of 100 nucleotides bears and added methyl group, which is
always attached to carbon 5 of cytosine in the 5’-CG-3’ rich island that
are often located in or near transcriptional regulatory regions.
DNA methylation serves more to maintain a gene in an inactive state
than as a mechanism for initial inactivation.
The extent of DNA methylation varies significantly among eukaryotes,
being strong in mammals and higher plants but rare in yeast and
Drosophila.
X-chromosome inactivation in female mammals.
The non-gamete cells of females each contain two copies of the X
chromosome, one inherited from each parent.
Early in development, one X chromosome in each existing cell is
randomly inactivated by condensation into a tight mass of
heterochromatin.
The inactivated X chromosome is strongly methylated and does not
participate in transcription initiation.
After X chromosome inactivation in embryonic cell - all cellular
descendants inactivate the same X chromosome.
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Methylation plays a role in most complex human diseases. Information derived from
methylation but not from any of the known genomic methods are:
•
The methylation status of a gene informs us about its current and its past
activation status
•
The methylation of the promoter of a gene can provide information as to how
easily a promoter can be activated
Methylation patterns are not only different between the tissues of one
individual, but - as known from animal studies - between different
populations
•
•
The overall methylation level of a cell`s DNA strongly influences genome
stability, allowing the prediction of malignancy of tumor cells
•
As viral sequences are silenced by methylation, the activity of viruses may be
predicted and gene-therapeutic vectors be optimized
From Epigenomics Inc.
Selected interactions in gene
silencing that involve DNA
methylation. Inverted repeats
(marked by an inverted set of
arrows) could trigger
methylation either by production
of transcripts (represented by
wavy lanes) that can form
dsRNA or by DNA–DNA
interactions. Methylation
(marked by lollipops) can
accumulate in the promoter or
coding regions of a gene.
Hypermethylation of a promoter
correlates with TGS. Increased
levels of methylation in a coding
region (the open reading frame
[ORF]) may not have immediate
consequences.
From Paszkowski and
Whitman, 2001, Current Op
Plant Biol
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Selection of mutants. Primary
selection of mutant
candidates: hygromycinresistant seedling after 8 days
selection on 15 mg/l
hygromycin (a), Northern (b),
and Southern (c) blot analysis
of the parental line A with the
silent hpt gene and the
mutants som1-som8. Both
membranes were probed with
the hpt coding region.
Genomic DNA was digested
with BamHI.
From Mittelsten-Scheid et al.,
PNAS, 1998
Methylation analysis. The
same Southern blot of DNA
cut with HpaII was probed
with the hpt coding region
(a), or with a fragment
containing the centromeric
180-bp repeat. Lanes:
1, pGl2 plasmid used to
generate the parental line A;
2, wt; 3, parental line A; 411, som 1-8, 12, mutant
ddm1/ddm1 with reduced
methylation (20);
13, ddm1/A; 14, METIas T3
#10.5 transgenic for a
methyltransferase antisense
construct (21); 15, METIas
T3 #10.5/A
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Effects of histones on transcription
•Core histones:
H2A, H2B, H3 and H4 had caused a mild repression of
genetic activity.
•TFs had no effect of this repression
•Core +H1 – strong repression of genetic activity
•This repression could be blocked by activators
In vitro transcription of reconstituted chromatin
Set-up:
•Plasmid DNA with Drosophila Krueppel gene
•Core histones at various ratios
•Polyglutamate used as a vehicle to to help histones deposit on
DNA
•Primer extension was performed to measure transcription
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Outcome:
•core histone inhibit the transcription is a dose-dependent
manner.
•Transcription of reconstituted chromatin with av. Of one
nucleosome per 200 bp exhibits 75% repression relative to
naked DNA.
•Remaining 25% is due to promoter sites not covered by
nucleosome cores.
•Activators, like Sp1, can not counteract the repression caused
by nucleosome core formation
Why 75% repression?
Nucleosomes slow the polymerase?
Or 25% of promoters might be left free ?
Experiment to check this:
Hypothesis: if a promoter is not blocked by nucleosomes and is
available for polymerase, it should also be available for the
restriction enzyme.
Gene has XbaI site downstream of the start site, this enzyme
should cut at the genes not protected by nucleosomes. This
would render gene inactive.
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Histone H1
Role-nucleosome cross-linker
Addition of it adds to inhibition of transcription caused by core
histones
This repression can be counteracted by transcription factors
Some TFs –Sp1, GAL4 act as antirepressors –prevent repression
by H1 and as transcription activators.
Some – like GAGA factor – are just antirepressors. They may
compete with H1 for binding sit5es of DNA template.
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Nucleosome positioning
Nucleosome –free zones – there
is evidence of the nucleosomefree zones in control regions of
active genes.
Active genes tend to have
DNAse-hypersensitive control
regions – this is at least due to
the absence of nucleosomes
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•Some active genes have an array of precisely positioned
nucleosomes in their control regions
•The same control regions, in tissues where these genes are not
active, do not have positioned nucleosomes
•The positioned nucleosomes can overlap the binding sites for
activators
•The activators and nucleosomes can coexist in the same place.
•Activators have a role in positioning nucleosomes
Histone acetylation
•Occurs in cytoplasm and in nucleus
•HAT B enzyme in cytoplasm prepares histones for incorporation
into nucleosomes
•Acetyl groups are removed in the nucleus
•Nuclear acetylation – HAT A
•Correlates with transcription activation
•A variety of activator have HAT A activity, allowing them to
loosen the association of nucleosomes with the gene’s control
region, thereby enhancing transcription
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•
Histone deacetylation
Deacetylation – repression event
•
Known transcription repressors, such as nuclear receptors
w/o ligands, interact with corepressors, which interact with
histone deacetylases.
•
DATs remove acetyl groups from histone tails, tightening
the grip of nucleosomes and DNA
A heterodimer of retinoic acid receptor and retinoic acid receptor X
binds to an enhancer.
In the absence of ligand - RA – the receptor binds to co-repressor,
which binds histone deacetylase (HDAC1)
HDAC1 removes acetyl groups –tight association with DNA.
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Activation and repression by
the same nuclear receptor:
Normal: No receptor dimer
is bound to hormone
response element.
Histones moderately
acetylated.
Repressed:
Receptor bound to TRE w/o
hormone. It interacts with
corepressors, they interact
with deacetylase.
Active:
receptor, hormone,
coactivators All activators
have the HAT A activity
Chromatin remodelling:
Swi/Snf family – disrupt the core histones of nucleosomes
Iswi family – move nucleosomes
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Reading:
p. 386-421.
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