Chromatin
Chromatin structure
Eukaryotic DNA is:
–
In a complex 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
¾ Genome packaging: Chromosomes - DNA and
associated proteins, which together are called chromatin.
¾ Two types of proteins in chromatin: histones and
nonhistone proteins.
¾ Nonhistone proteins: diverse structural, enzymatic, and
regulatory proteins.
¾ Histones: Packaging of eukaryotic DNA depends on
histones.
9 Approx. 10% of the chromatin remains in a condensed,
compacted form throughout interphase. This compacted
chromatin is seen at the periphery of the nucleus.
Two Basic States of Chromatin
Heterochromatin:
– Darkly stained, relatively condensed, genetically inactive
– 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.) (ex.
inactive X chromosome).
– All DNA (chromatin) is heterochromatic during mitosis.
Constitutive heterochromatin (CH)
– remains in the compacted state in all cells at all times
(DNA that is permanently silenced). The bulk of the CH is
found in and around the centromere of each chromosome
in mammals.
– The DNA of CH consists primarily of highly repeated
sequences and contains relatively few genes.
– When genes that are normally active are transposed
into a position adjacent to CH, they tend to become
inactivated.
Facultative heterochromatin (FH)
– It 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.
Two Basic States of Chromatin
Euchromatin:
– Stains more lightly, less condensed.
– Capable of genetic activity (transcription). It is a normal
state of the most DNA during interphase.
– Euchromatin is often divided into several distinguishable
states of folding
• tightness of folding is probably really continuous from
relatively loose to relatively tight).
• correlation between folding and function is complex.
Histones
• Eukaryotic cells contain 5
kinds of histones
–
–
–
–
–
H1 (lys rich)
H2A (slightly lys rich)
H2B (slightly lys rich)
H3
H4 (arg rich)
• Each histone type isn’t
homogenous
– Gene reiteration
– Posttranslational
modification (ex.
acetylation)
Source: Panyim and Chalkley, Arch. Biochem.
& Biophys. 130, 1969, f. 6A, p.343.
Properties of Histones
• Abundant proteins whose mass in nuclei nearly
equals that of DNA
• Pronounced positive charge at neutral pH
• Most are well-conserved from one species to
another (ex. H4; H3)
• Not single copy genes, repeated many times (10
to 100 copies per genome)
– Some copies are identical
– Others are quite different
– H4 has only had 2 variants ever reported
Nucleosomes
• Chromosomes are long, thin molecules that will
tangle if not carefully folded
• Folding occurs in several ways
• First order of folding is the nucleosome
– X-ray diffraction has shown strong repeats of structure
at 100Å intervals
– This spacing approximates the nucleosome spaced at
110Å intervals
Histones in the Nucleosome
• Chemical cross-linking in solution:
– H3 to H4
– H2A to H2B
• H3 and H4 exist as a tetramer (H3-H4)2
• Concentration of H1 is half of the other histones
• Cutting chromatin with nucleases yeilds 200bp
fragments
• Repeated structure of chromatin is composed of
– 1 histone octamer per 200 bp of DNA
– Octamer composed of:
• 2 each H2A, H2B, H3, H4
• 1 each H1
H1 and Chromatin
• Treatment of chromatin with trypsin or high salt
buffer removes histone H1
• This treatment leaves chromatin looking like
“beads-on-a-string”
• The beads were named nucleosomes
– Core histones form a ball with DNA wrapped around
the outside
– DNA on outside minimizes amount of DNA bending
– H1 also lies on the outside of the nucleosome
Nucleosomes
Nucleosome Structure
• Central (H3-H4)2 core attached to H2A-H2B dimers
• Grooves on surface define a left-hand helical ramp –
a path for DNA winding
– DNA (about 147bp) winds almost twice around the
histone core condensing DNA length by 6- to 7-X
– Core histones contain a histone fold:
• 3 α-helices linked by 2 loops
• Extended tail of abut 28% of core histone mass
• Tails are unstructured
• Histone H1 is more easier removed from a chromatin
and is not a part of the core nucleosome
Histones in Nucleosome
H2A – H2B dimer – dark blue;
(H3-H4)2 tetrame light blue
H2A – yellow; H2B – red; H3
– blue, H4 - green
The 30-nm Fiber
• Second order of chromatin folding produces a
fiber 30 nm in diameter
– The string of nucleosomes condenses to form the
30-nm fiber in a solution of increasing ionic
strength
– This condensation results in another six- to sevenfold condensation of the nucleosome itself
• Four nucleosomes condensing into the 30-nm
fiber form a zig-zag structure
Formation of the 30-nm Fiber
• Two stacks of nucleosomes form a lefthanded helix
– Two helices of polynucleosomes
– Zig-zags of linker DNA
• Role of histone H1
– 30-nm fiber can’t form without H1
– H1 crosslinks to other H1 more often than to
core histones
Structure of the 30-nm Fiber
Higher Order Chromatin Folding
• 30-nm fibers account
for most of chromatin in
a typical interphase
nucleus
• Further folding is
required in structures
such as the mitotic
chromosomes
• Model favored for such
higher order folding is a
series of radial loops
(35-85bp long)
Source: Adapted from Marsden, M.P.F. and U.K.
Laemmli, Metaphase chromosome structure:
Evidence of a radial loop model. Cell 17:856,
1979.
Higher Order Chromatin Folding
Chromatin Structure and Gene
Activity
• Histones, especially H1, have a repressive effect on
gene activity in vitro
• Two families of 5S rRNA genes studied are oocyte and
somatic genes
– Oocyte genes are expressed only in oocytes
– Somatic genes are expressed both in oocytes and
somatic cells
– Somatic genes form more stable complexes with
transcription factors
– Formation of stable complex of nucleosome with gene
control region (H1 is required) prevents transcription
factors from interacting with the gene and represses
transcription
Transcription Factors and Histones
Control the 5S rRNA
• Gene activation by TFIIIs
prevented formation of
stable nucleosome
complexes with internal
control region
• Stable complexes require
histone H1 and exclude
TFIIIs once formed, so
that genes are repressed
Effects of Histones on Transcription of
Class II Genes
• Core histones assemble nucleosome cores on naked DNA
• Transcription of reconstituted chromatin with an average of 1
nucleosome / 200 bp DNA exhibits 75% repression relative to
naked DNA
– RNA polymerase was slowed down by 75% ???
– 75% of RNA pol was completely blocked by nucleosomes. Only
25% of promoter was available ???
• Remaining 25% of transcription can be eliminated by cutting
chromatin with restriction enzyme that cuts just downstream
from transcription start site
¾ Remaining 25% activity is due to promoter sites not
covered by nucleosome cores
Histone H1 and Transcription
• Histone H1 causes further repression of template activity,
in addition to that of core histones
• H1 repression can be counteracted by transcription
factors
• Sp1 and GAL4 act as both:
– Antirepressors preventing histone repressions
– Transcription activators
• GAGA factor:
– Binds to GA-rich sequences in the Krüppel promoter
– It is only an antirepressor – prevents repression by
histones
Model of Transcriptional Activation
Source: Adapted from Laybourn, P.J. and J. T. Kadonaga, Role of nucleosomal cores and histone H1 in
regulation of transcription by polymerase II. Science 254:243, 1991.
Nucleosome Positioning
• Model of activation and antirepression asserts
that transcription factors can cause antirepression
by:
– Removing nucleosomes that obscure the promoter
– Preventing initial nucleosome binding to the promoter
• Both actions are forms of nucleosome positioning:
activators force nucleosomes to take up positions
around, not within, promoters
Nucleosome-Free Zones
• Nucleosome positioning would result in
nucleosome-free zones in the control regions of
active genes
• Assessment in a circular chromosome can be
difficult without some type of marker
Detecting DNase-Hypersensitive
Regions
• Active genes tend to have DNase-hypersensitive
control regions
• Part of this hypersensitivity is due to absence of
nucleosomes
Histone Acetylation
• Histone acetylation occurs in both cytoplasm and nucleus
• Cytoplasmic acetylation carried out by HAT B (histone
acetyltransferase B)
– Prepares histones for incorporation into nucleosomes
– Acetyl groups later removed in nucleus
• Nuclear acetylation of core histone N-terminal tails
– Catalyzed by HAT A
– Correlates with transcription activation
– Number of coactivators have a HAT A activity that allows
loosening of association between nucleosomes and gene’s control
region
– Acetylation attracts bromodomain proteins (TAFII250), essential
for transcription
Histone Deacetylation
• Transcription repressors bind to DNA sites and
interact with corepressors, which in turn bind to
histone deacetylases
– Repressors
• Unliganded nuclear receptors
• Mad-Max (mammals) (Myc-Mad and Mad-Max
dimers)
– Corepressors
• NCoR/SMRT (mammals)
• SIN3 (yeast)
– Histone deacetylases - HDAC1 and 2
Ternary Protein Complexes
• Assembly of complex brings
histone deacetylases close
to nucleosomes
• Deacetylation of core
histones allows
– Histone basic tails to bind
strongly to DNA and to
histones in neighboring
nucleosomes (ex. N-tail H4)
– This inhibits transcription
Activation and Repression (Type II Nuclear Receptor)
Source: Adapted from Wolfe, A.P., 1997. Sinful repression. Nature 387:16-17.
¾ Deacetylation of core histones removes binding
sites for bromodomain proteins that are essential for
transcription activation
Chromatin Remodeling
• Activation of many eukaryotic genes requires
chromatin remodeling
• Several protein complexes carry this out
– All have ATPase harvesting energy from ATP
hydrolysis for use in remodeling
– Remodeling complexes are distinguished by
ATPase component
• SWI/SNF
• ISWI
• NuRD
• INO80
Remodeling Complexes
• SWI/SNF
– In mammals, has BRG1 as ATPase
– 9-12 BRG1-associated factors (BAFs)
• A highly conserved BAF is called BAF 155 or 170
• Has a SANT domain responsible for histone
binding
• This helps SWI/SNF bind nucleosomes
• ISWI
– Have two SANT domains (one is involved in
histone binding (acidic); one is involved in DNA
binding (positively charged) – the SLIDE domain)
SWI/SNF Chromatin Remodeling
Mechanism of Chromatin Remodeling
• Mechanism of chromatin remodeling involves:
– Mobilization of nucleosomes
– Loosening of association between DNA and core
histones
• Catalyzed remodeling of nucleosomes involves
formation of distinct conformations of nucleosomal
DNA/core histones when contrasted with:
– Uncatalyzed DNA exposure in nucleosomes
– Simple nucleosome sliding along a DNA stretch
Remodeling in Yeast HO Gene Activation
• Chromatin immunoprecipitation (ChIP) can reveal
the order of binding of factors to a gene during
activation
• As HO gene is activated:
– First factor to bind is Swi5
– Followed by SWI/SNF and SAGA containing HAT Gcn5p
– HAT Gcn5p recruits the activator SBF
– Next general transcription factors and other proteins bind
• Chromatin remodeling is among the first steps in
activation of this gene
• Order could be different in other genes (HAT may bind
before the SWI/SNF complex)
Chromatin Immunoprecipitation
The Histone Code
The Histone Code:
– The combination of histone modifications on a given
nucleosome near a gene’s control region affects
efficiency of that gene’s transcription
– This code is epigenetic, not affecting the base
sequence of DNA itself
Histone modifications include:
– Acetylation
– Methylation
– Phosphorylation
– Ubiquitylation
– Sumoylation
Remodeling in the Human IFN-b
• Activators in the IFN-β enhanceosome can recruit a
HAT (GCN5), the SWI/SNF complex and GTFs
– HAT acetylates some Lys on H3 and H4 in a nucleosome
at the promoter
– Protein kinase phosphorylates Ser on H3
– This permits acetylation of another Lys on H3
• Acetylation attracts the CBP-RNA pol II holoenzyme via
bromodomains in CBP
• SWI/SNF complex in holoenzyme loosens association
between promoter DNA and nucleosome
Remodeling in the Human IFN-β
• Remodeling allows TFIID to bind 2 acetylated
Lys in the nucleosomes through the dual
bromodomain in TAFII250
• TFIID binding
– Bends the DNA
– Moves remodeled nucleosome aside
– Paves the way for transcription to begin
Model for Histone Code
– Information flows from enhancer to
nucleosome
– Enhanceosome assembles at the
promoter
– Activators recruit GCN5 (HAT) that
acetylates lysine K8H4 and K9H3
– Enhanceosome recruits a protein
kinase that phosphorylates serine S10H3,
allowing GCN5 to acetylate lysine K14H3,
therefore completing the histone code.
Model for Histone Code
– Acetylated lysine K8H4 attracts the
SWI/SNF complex that remodels the
nucleosome
– Remodeled nucleosome permits
binding the TFIID
– TFIID is attracted by both TATA box
and acetylated lysines K9H3 and
K14H3
– TFIID bends DNA and moves
remodeled nucleosome 36bp
downstream
– Transcription begins
Heterochromatin
• Euchromatin: relatively extended and
open chromatin that is potentially active
• Heterochromatin: very condensed with its
DNA inaccessible
– Microscopically appears as clumps in higher
eukaryotes
– Repressive character able to silence genes as
much as 3 kb away
Hetero- and Euchromatin
Heterochromatin and Silencing
• Formation of at tips of yeast chromosomes
(telomeres) with silencing of the genes is the
telomere position effect (TPE)
• Depends on binding of proteins
– RAP1 binds to telomeric DNA (C1 – 3A sequence)
– Recruitment of proteins in this order:
• SIR3
• SIR4
• SIR2
SIR Proteins
• Heterochromatin at other locations in
chromosome also depends on the SIR proteins
• SIR3 and SIR4 interact directly with histones H3
and H4 in nucleosomes
– Acetylation of Lys 16 on H4 in nucleosomes
prevents interaction with SIR3
– Blocks heterochromatin formation
• Histone acetylation also works in this way to
promote gene activity
Heterochromatin and Silencing
Histone Methylation
• Methylation of Lys 9 in N-terminal tail of H3
attracts HP1 (HMTase associated protein)
• This recruits a histone methyltransferase
(HMTase)
– Methylates Lys 9 on a neighboring nucleosome
– Propagates the repressed, heterochromatic state
• Methylation of Lys and Arg side chains in core
histones can have either repressive or activating
(ex. K4H3) effects
Histone Methylation
Modification Interactions
• The modifications
shown above the tail
are activating
– Ser phosphorylation
– Lys acetylation
• Modification below
the tail (Lys
methylations) is
repressive
Modification Combinations
• Methylations occur in a given nucleosome in
combination with other histone modifications:
– Acetylations
– Phosphorylations
– Ubiquitylations
• Each particular combination can send a different
message to the cell about activation or repression
of transcription
• One histone modification can also influence other,
nearby modifications
Nucleosomes and Transcription Elongation
• An important transcription elongation facilitator is FACT
(facilitates chromatin transcription)
– Composed of 2 subunits:
• Spt16
– Binds to H2A-H2B dimers and destabilizes nucleosome
– Has acid-rich C-terminus essential for these nucleosome
remodeling activities
• SSRP1 binds to H3-H4 tetramers
– Facilitates transcription through a nucleosome by promoting loss
of at least one H2A-H2B dimer from the nucleosome
• FACT also acts as a histone chaperone promoting readdition of H2A-H2B dimer to a nucleosome that has lost
such a dimer
Reading:
R. Weaver, Molecular Biology, 4th ed.
Chapter 12: pages 321-358.
w/o details of figures: 13.14,13.15,13.22,13.25,13.30,13.31,
13.37, 13.40.