Heterochromatin

1
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
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 6-33
2
Histone Modifications Associated with
Heterochromatin and Euchromatin
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 6-33
3
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
4
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)
5
Heterochromatin Functions
DNA or H3 methylation recruits adaptors such as
HP1
Adaptors recruit effectors that are involved in
chromosome segregation, gene silencing,
transcriptional activation, and histone
modification
from Grewal and Gia, Nature Rev.Genet. 8, 35
(2007)
6
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)
7
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)
8
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)
9
Mechanism of Silencing at Telomeres
Sir2 deacetylates histones
Sir3,4 binds deacetylated histones and recruits
additional Sir2
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 7-35
10
Insulators Prevent the Progression of Condensed
Chromatin
Insulators protect genes from inappropriate
signals
Insulators block the action of distal enhancers
Insulators prevent the spreading of
heterochromatin
from West et al, Genes Dev. 16, 271 (2002)
11
gypsy Retrotransposon Contains an Insulator
gypsy protects a transgene from position effects
su(Hw) is necessary for enhancer blocking activity
gypsy contains a su(Hw) binding site
su(Hw) blocks the process that brings enhancer
and promoter together
Formation of insulator bodies at the nuclear
periphery to divide the chromosome into looped
domains
Multiple su(Hw) binding sites can inhibit
enhancer blocking activity
12
Models for Heterochromatin Barrier Formation
Stable block interrupts propagation of
heterochromatin
Active barrier recruits a complex containing
chromatin remodeling activity
from Donze and Kamakaka, BioEssays 24, 344 (2002)
13
Epigenetics
Heritable changes in gene function that cannot be
explained by changes in gene sequences
DNA methylation
Polycomb and trithorax complexes
14
Epigenetic Modifications Within an Arabidopsis
Chromosome
Heterochromatin correlates with epigenetic marks
from Zhang, Science 320, 489 (2008)
15
DNA Methylation
Methylation at CpG residues
Sites of methylation
Inactive X
Imprinted loci
Transposon-derived sequences
CpG islands
Methylation patterns are reproduced at each round
of cell division
16
Establishment of DNA Methylation Pattern
Most CpGs are unmethylated before implantation
RNA pol II recruits H3K4 methyltransferase
DNMT3L only binds unmethylated H3K4 and recruits
DNA methyltransferases
from Cedar and Bergman, Nature Rev.Genet. 10, 295
(2009)
17
Mechanisms for Repression Mediated by MBD Proteins
from Wade, BioEssays 23, 1131 (2001)
18
MeCP2 Regulates Gene Expression in Response to
Neural Activity
Rett Syndrome is linked to mutations in MECP2 on
the X chromosome
MeCP2 binds CpG residues and silences target
genes such as BDNF and corticotropin-releasing
hormone
from Bienvenu and Chelly, Nature Rev.Genet. 7,
415 (2006)
Neural activity triggers MeCP2 phosphorylation
and target gene activation
Hippocampal neurons grow dendrites with fewer
branches when MeCP2 is blocked
from Miller, Science 314, 1356 (2006)
19
Establishment of Cell Identity in Drosophila
Embryos
Segment identity is established by sequential
spatially-localized expression of specific genes
Regulatory genes are expressed transiently
Transcriptional memory is maintained throughout
development
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 15-24
20
Misexpression of Homeotic Genes Lead to
Morphological Abominations
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 15-25
21
Polycomb and Trithorax Complexes
Prevents changes in cell identity by preserving
transcription patterns
Chromatin is altered in a heritable manner
Polycomb-group Proteins
Maintains a silenced state
Prevents chromatin remodelling
Trithorax-group Proteins
Maintains an active state
Counteracts the action of PcG proteins
Memory system composed of PcG and trxG complexes
is linked to the histone code
22
Model for PcG Formation and Function
PcG complexes are recruited to PREs
PRC2 complex methylates H3 K9 and K27
H3K27me3 recruits Polycomb and PRC1 complex
H3K27me3 is segregated to both daughter DNAs to
maintain repression
from Lund and van Lohuizen, Curr.Opin.Cell Biol.
16, 239 (2004)
23
Demethylation of H3K27me3 Promotes Gene Activation
PRC2 is recruited to H3K27me3 to mediate gene
repression
UTX and JMJD3 are recruited to Hox promoters and
reverse repression
Change in cell fate is mediated by H3K27
demethylation and H3K4 methylation, whose
activities are present in the same complex
from Rivenbark and Strahl, Science 318, 403 (2007)
24
Trithorax Complex Mechanism of Action
TrxC methylates H3K4 and recruits HAT and
remodeling complexes
Acetylated H3K9 prevents methylation, and
prevents HP1 binding
25
Somatic Cell Reprogramming
Pleuripotency genes in somatic cells have
methylated CpG islands
Epigenetic marks must be reset to generate
induced pleuripotent stem (iPS) cells
Repressive histone methylation marks must be
removed, followed by removal of DNA methylation
which activates the gene
from Cedar and Bergman, Nature Rev.Genet. 10, 295
(2009)
26
Co-suppression
Increase in gene copy number results in
decreased expression
Dependent on PcG genes
PcG complexes interact in trans
from Pirrotta, Cell 93, 333 (1998)
27
Imprinting
Expression of only one allele of a locus
Only 80 genes in mammals are imprinted
Most imprinted genes are involved in growth
control
Imprinted genes involves allele specific
methylation and is resistant to genome-wide
demethylation
Clusters of imprinted genes contain noncoding
RNAs that are involved control allele-specific
expression
28
Imprinted Expression of the H19 and Igf2 Genes
Maternal H19 expression
Paternal Igf2 expression
from Reik and Murrell, Nature 405, 408 (2000)
29
Imprinting is Regulated by a Methylation-sensitive
Boundary
ICR is methylated in the male germ line
ICR is protected from methylation in the female
germ line by CTCF
CTCF binding to the ICR in females prevents
activation of Igf2 by downstream enhancer
In males, the downstream enhancer activates Igf2
and H19 expression is repressed by DNA
methylation
from Reik and Murrell, Nature 405, 408 (2000)
30
Imprinting of the PWS-AS Locus
from Ferguson-Smith and Surani, Science 293, 1086
(2001)
The AS-ICR is required for methylation and
inactivation of the PWS-ICR in females to
repress nearby genes
The AS-ICR is nonfunctional in males allowing
the PWS-ICR to activate nearby genes
The PWS-ICR promotes expression of an antisense
Ube3a transcript in males
31
Dosage Compensation Mechanisms
Genomes compensate for different numbers of sex
chromosomes by adjusting gene expression levels
from Straub and Becker, Nature Rev.Genet. 8, 47
(2007)
32
X Inactivation in Mammals
X inactivation is initiated from the Xic
Xist and Tsix partially overlap and are
transcribed in opposite directions from the Xic
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 22-7
33
Model for the Initiation of X Inactivation
The Xic in female cells colocalize prior to X
inactivation
Low expression of Tsix from Xi leads to Xist
transcription
Xist RNA coats the Xi in cis
The chromatin structure of Xi becomes condensed
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 22-7
34
Stepwise Progression of X Inactivation in
Differentiating ES Cells
One X chromosome is converted to facultative
heterochromatin
Xist transcription off the inactive X initiates
chromatin modification events
X inactivation is maintained epigenetically
from Brockdorff, Trends Genet. 18, 352 (2002)
35
Calico Cats
One of the genes controlling fur color is on the
X chromosome
B orange b - black
Female mammals are genetic mosaics
Random X inactivation early in embryonic
development leads to patchworks of skin cells
expressing each allele
36
The Dosage Compensation Complex in Drosophila
SXL in females prevents MSL2 translation
MSL2 in males stabilizes roX, MSL1, and MSL3
DCC binds to high affinity sites on X chromosome
DCC spreads to nearby sites on active chromatin
from Gilfillan et al., FEBS Lett. 567, 8 (2004)
H4K16 acetylation impedes formation of condensed
chromatin structure
37
DCC is Localized to the X Chromosome
DCC localization is determined by staining of
polytene chromosomes with anti-MSL1
DCC associates almost exclusively with
transcribed regions
from Straub and Becker, Nature Rev.Genet. 8, 47
(2007)