Chromatin Structure

1
Chromatin Structure
Decondensation of the 30 nm fiber produces beads
on a string
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 10-19
2
The Nature of the Nucleosome
from Alberts et al., 3rd ed., Fig. 8-10
3
Nucleosomes are Packaged into a 30 nm Fiber
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 10-21
4
Packing of Chromatin
Scaffold-associated regions can act as boundaries
Condensed chromosomes are visible during
metaphase
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 10-24
5
Nucleosomal Histones and Their Variants
from Sarma and Reinberg, Nature Rev.Mol.Cell
Biol. 6, 139 (2005)
6
Genes Can be Localized on Drosophila Polytene
Chromosomes
Polytene chromosomes exhibit a characteristic
banding pattern
Localization of a gene by in situ hybridization
Biotinylated probe was detected by avidin
conjugated to alkaline phosphatase
AP substrate results in the formation of an
insoluble precipitate at the site of
hybridization
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 10-30
7
Chromosome Puffs
Ecdysone produces a characteristic pattern of
puffs in polytene chromosomes of salivary glands
Puffs correspond to actively transcribed genes
from Alberts et al., 3rd ed., Fig. 8-23
8
Michael Ashburner
9
The Ashburner Model for Ecdysone Action
Culture larval salivary glands with ecdysone and
observe the same chromosome puffing pattern as
in vivo
Cyclohexamide prevents regression of early puffs
and induction of late puffs
This model was proposed in 1974 by observation
of chromatin structure
from Thummel, Insect Biochem.Mol.Biol. 32, 113
(2002)
10
Action of HMG-box Proteins
HMG-box proteins bend DNA
DNA bending can affect transcription and
site-specific recombination
from Thomas and Travers, Trends Bioch.Sci. 26,
167 (2001)
11
Transcriptionally Active Genes are More
Susceptible to DNase I Digestion
Loss of 4.6 kb Bam HI fragment when the b-globin
gene is active and histones are acetylated
The 4.6 kb Bam HI fragment is present when the
b-globin gene is inactive and histones are
deacetylated
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 10-22
12
Chromatin Immunoprecipitation (ChIP)
Use antibody to acetylated histone tail to
determine the acetylation state of chromatin
Antibody against any DNA binding protein
determines the location of the binding site
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 11-31
13
Histone Modifications
Posttranslational modifications and histone
variants contribute to structural and functional
characteristics of chromatin
Chromatin structure is a source of epigenetic
information
Histone code defines actual or potential
transcription states
14
Histone are Modified at Multiple Positions
Each modification has a unique biological role
Histone modifications are interdependent
from Turner, Cell 111, 285 (2002)
15
Properties of Acetylated Histones
Less positively charged
Chromatin is less condensed
16
Control of Gene Expression by Acetylation
Repressor recruits a complex that contains a
histone deacetylase
Activator recruits a complex that contains a
histone acetylase
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 11-32
17
Acetylation does not always correlate with
increased transcription
Depends on the particular lysine being acetylated
Euchromatin H4 is acetylated at K5 and
K8 Heterochromatin H4 is acetylated at
K12 Hyperactive male X chromosome H4 is
acetylated at K16
18
Histone Acetylation is Involved in Cancer
pRb interacts with HDAC Some viral oncoproteins
inhibit pRb-HDAC interaction
E1A binds to p300/CBP Regulates HAT activity
19
Effect of Histone H3 K9 Methylation
SUV39 methylates K9
Methylated K9 recruits HP1
Heterochromatin formation
HP1 binds to SUV39 to propagate methylation
Methylated K9 or phosphorylated S10 inhibits
methylation of K9
from Turner, Cell 111, 285 (2002)
20
Effect of Histone H3 K4 Methylation
Set9 methylates K4
Inhibits association of NuRD remodeling and
deacetylase complex
Inhibits association of SUV39
Methylated K4 is associated with active genes
from Turner, Cell 111, 285 (2002)
21
The Effect of Histone H3 K9 Methylation Depends
on Other Histone Modifications
Drosophila ASH1 methylates H3 on K4 and K9 and
H4 on K20
Facilitates Brahma binding
Inhibits HP1 binding
In this context, methylated K9 is not a
determinant of silencing
from Turner, Cell 111, 285 (2002)
22
Aging in Yeast
Mother cell has a finite lifespan
Homologous recombination of rDNA locus generates
rDNA circles
ERCs accumulate in mother cells and cause aging
Inhibition of ERC formation extends longevity
23
Role of Sir2 in Aging
Sir complex promotes formation of repressive
chromatin structure
Sir2 is a NAD-dependent HDAC
HDAC activity represses ERC formation
Loss of silencing in aging cells
Sir2 mutants have shorter lifespans
24
Caloric Restriction Increases Sir2 Activity
Caloric restriction increases longevity
Extended lifespan is dependent on Sir2
Decreased ERC formation during caloric restriction
Increased NAD during caloric restriction
regulates Sir2 function
Sir2 may be the sensor of oxidation state of
cells to coordinate the pace of aging
Sir2 is recruited to sites of DNA damage to
maintain integrity of silenced chromatin
ERCs are not linked to aging in humans
Loss of silencing and misregulated transcription
may be a cause of aging
25
Stress-activated Deacetylation of p53 Leads to
Cell Survival
Senescence is a tumor protection mechanism
induced by cellular stresses
SIRT1 may modulate proper activity level of p53
SIRT1 promotes p53 deacetylation leading to cell
survival
from Smith, Trends Cell Biol. 12, 404 (2002)
26
Heterochromatin
Darkly stained and condensed
Transcriptionally silent and silences adjacent
genes
Present at centromeres and telomeres
HP1 interacts with H3 only when K9 is methylated
Repressive structure can be propagated
Euchromatic gene placed in heterochromatin is
repressed
27
Initiation of Heterochromatin Assembly
from Grewal and Gia, Nature Rev.Genet. 8, 35
(2007)
Transcription factors and RNAi machinery bind to
specific sequences or repetitive elements to
recruit histone modifying enzymes
Modified histones recruit HP1
HP1 recruits histone modifying enzymes to
facilitate heterochromatin spread
Boundary elements prevent further heterochromatin
spread
28
Propagation of Heterochromatin
Passage of the replication fork releases
parental modified nucleosomes
Nucleosome binding sites are created by
recruitment of CAF1 by PCNA
CAF1-bound HP1 recruits Suv39h, Dnmt1, and HDAC
Methylated histones provide new HP1 binding sites
Structural RNA associates
from Maison and Almounzi, Nature Rev.Mol.Cell
Biol. 5, 296 (2004)
29
Role of RNAi in Heterochromatin Formation in S.
pombe
dsRNA is transcribed from centromeric repeats or
synthetic hairpin RNAs
dsRNA is processed to siRNA
siRNA promotes H3 K9 methylation by Clr4
Methylated H3 K9 recruits Swi6 to form silenced
chromatin
Transcription of the top strand of centromeric
repeats is repressed
Rdp1 activity ensures continuous dsRNA synthesis
Recruitment of Clr4 by Swi6 chromatin leads to
spread of heterochromatin
from Schramke and Allshire, Science 301, 1069
(2003)
30
Histone Acetylation Regulates SIR Complex
Association with Chromatin
Acetylation of histone H4 K12 facilitates
binding of Sir complex if K16 is deacetylated
from Grunstein, Cell 93, 325 (1998)
31
Formation of Telomeric Heterochromatin
RAP1 binds to C1-3A repeats
Recruits Sir proteins
Overexpression of Sir3 causes spread of
telomeric heterochromatin
Silencing decreases exponentially with distance
from Grunstein, Cell 93, 325 (1998)