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

Histone Modifications Associated with Heterochromatin and Euchromatin

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-33

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

Boundary elements prevent further heterochromatin spread

HP1 recruits histone modifying enzymes to facilitate heterochromatin spread

Modified histones recruit HP1

from Bannister et al., Nature 410, 120 (2001)

Mechanism of Heterochromatin Spreading

HP1 binds to H3K9me3

HP1 recruits SUV39H1 methylase

SUV39H1 methylates H3K9 on neighboring nucleosomes

Heterochromatin spreading is restricted by boundary elements

Propagation of Heterochromatin

from Maison and Almounzi, Nature Rev.Mol.Cell Biol. 5, 296 (2004)

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 Grewal and Gia, Nature Rev.Genet. 8, 35 (2007)

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

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 heterochromatinfrom Schramke and Allshire, Science 301, 1069 (2003)

Formation of Telomeric Heterochromatin

from Grunstein, Cell 93, 325 (1998)

RAP1 binds to C1-3A repeats

Recruits Sir proteins

Overexpression of Sir3 causes spread of telomeric heterochromatin

Silencing decreases exponentially with distance

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

Insulators Prevent the Progression of Condensed Chromatin

from West et al, Genes Dev. 16, 271 (2002)

Insulators protect genes from inappropriate signals

Insulators block the action of distal enhancers

Insulators prevent the spreading of heterochromatin

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

Models for Heterochromatin Barrier Formation

from Donze and Kamakaka, BioEssays 24, 344 (2002)

Stable block interrupts propagation of heterochromatin

Active barrier recruits a complex containing chromatin remodeling activity

Epigenetics

Heritable changes in gene function that cannot be explained by changes in gene sequences

DNA methylation

Nucleosome positioning

Histone variants and modifications

Epigenetic Modifications During Development

Epigenetically imposed restrictions to plasticity are erased in the germ line

Early mammalian development is characterized by progressive restriction of cellular plasticity accompanied by acquisition of epigenetic modifications

Epigenetic modifications impose a cellular memory that accompanies and enables stable differentiation

from Zhang, Science 320, 489 (2008)

Epigenetic Modifications Within an Arabidopsis Chromosome

Heterochromatin correlates with epigenetic marks

DNA Methylation

Methylation at CpG residues

Sites of methylation

Inactive X

Imprinted loci

Transposon-derived sequences

CpG islands and CpG island shores

Methylation patterns are reproduced at each round of cell division

Methylated CpG Islands Inhibit Transcription

Promoters are usually unmethylated

More than half of human promoters contain CpG islands

Methylated DNA recruits methyl-CpG-binding domain proteins which recruit histone modifying and chromatin-remodelling complexes

Unmethylated CpG islands recruit Cfp1 which associates with a histone methyltransferase creating H3K4me3

from Portela and Esteller, Nature Biotechnol. 28, 1057 (2010)

Methylated CpG Islands Inhibit Transcription

Promoters are usually unmethylated

More than half of human promoters contain CpG islands

Methylated DNA recruits methyl-CpG-binding domain proteins which recruit histone modifying and chromatin-remodelling complexes

Unmethylated CpG islands recruit Cfp1 which associates with a histone methyltransferase creating H3K4me3

from Portela and Esteller, Nature Biotechnol. 28, 1057 (2010)

Methylation of Repetitive Sequences Stabilize Chromosomes

Unmethylated repetitive sequences cause reactivation of endoparasitic sequences

from Portela and Esteller, Nature Biotechnol. 28, 1057 (2010)

RNA-dependent DNA Methylation in Plants

Methylation occurs in transposons and repetitive elements

PolIV transcribes ssRNA which is converted to dsRNA by RDR2

siRNA is produced by DCL3 and loaded onto AGO4

PolV produces IGN transcripts and recruits AGO4

siRNA-IGN duplex is formed and recruits DRM2

from Law and Jacobsen, Nature Rev.Genet. 11, 204 (2010)

from Law and Jacobsen, Nature Rev.Genet. 11, 204 (2010)

De Novo DNA Methylation in Mammals

DNMT3L interacts with unmethylated H3K4

DNMT3A is recruited and activated and forms a tetrameric complex

Active sites are separated by 8-10 bp and methylates opposite DNA strands

Tetramer oligomerizes and results in 10 bp pattern of methylation on the same strand

from Cedar and Bergman, Nature Rev.Genet. 10, 295 (2009)

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

Propagation of DNA Methylation State

Newly synthesized methylated DNA is hemimethylated

NP95 links DNMT1to hemimethylated DNA

DNMT1 is a maintenance methyltransferase and binds PCNA

NP95 binds hemimethylated DNA

from Richly et al., BioEssays 32, 669 (2010)

Mechanisms for Repression Mediated by MBD Proteins

from Wade, BioEssays 23, 1131 (2001)

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

Neural activity triggers MeCP2 phosphorylation and target gene activation

MeCP2 Regulates Gene Expression in Response to Neural Activity

Hippocampal neurons grow dendrites with fewer branches when MeCP2 is blocked

from Miller, Science 314, 1356 (2006)

from Bienvenu and Chelly, Nature Rev.Genet. 7, 415 (2006)

Establishment of Cell Identity in Drosophila Embryos

from Lodish et al., Molecular Cell Biology, 5th ed. Fig 15-24

Segment identity is established by sequential spatially-localized expression of specific genes

Regulatory genes are expressed transiently

Transcriptional memory is maintained throughout development

Misexpression of Homeotic Genes Lead to Morphological Abominations

from Lodish et al., Molecular Cell Biology, 5th ed. Fig 15-25

Polycomb-group Proteins

Maintains a silenced state

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

Prevents changes in cell identity by preserving transcription patterns

Polycomb and Trithorax Complexes

Chromatin is altered in a heritable manner

Prevents chromatin remodelling

Model for PcG Formation and Function

from Lund and van Lohuizen, Curr.Opin.Cell Biol. 16, 239 (2004)

PRC2 complex methylates H3 K9 and K27

H3K27me3 recruits Polycomb and PRC1 complex

H3K27me3 is segregated to both daughter DNAs to maintain repression

PcG complexes are recruited to PREs

from Richly et al., BioEssays 32, 669 (2010)

Propagation of H3K27 Methylation

EED2 binds H3K27me3

EED2 binding stimulates PRC2 activity

EZH2 methylates H3K27

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)

Trithorax Complex Mechanism of Action

TrxC methylates H3K4 and recruits HAT and remodeling complexes

Acetylated H3K9 prevents methylation, and prevents HP1 binding

Somatic Cell Reprogramming

from Cedar and Bergman, Nature Rev.Genet. 10, 295 (2009)

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

Brg1, a SWI/SNF component, is activated by cardiac stress

Brg1 suppresses expression of a CKI to promote myocyte proliferation

Brg1 forms a complex with HDAC and PARP and triggers a shift from -myosin heavy chain expression to -myosin heavy chain expression

Epigenetics and Heart Failure

from Hang et al., Nature 466, 62 (2010)

Brg1 promotes reprogramming to an embryonic state of transcription

Epigenetic Modifications May Drive Cognitive Decline

from Sweatt, Science 328, 701 (2010)

Chromatin remodeling in the hippocampus is necessary for stabilizing long term memories

Aged mice have lower H4K12 acetylation

HDAC inhibitor restores H4K12 acetylation and improved memory function

SIRT1 lof causes Alzheimer’s-like phenotype

SIRT1 deacetylates RAR

SIRT1 lof results in decreased -secretase transcription and increased A production

SIRT1 lof causes decreased Notch pathway activity and decreased neuronal repair

SIRT1 Deacetylase and Alzheimer’s Disease

from Wolfe and Selkoe, Cell 142, 194 (2010)

Prion Epigenetics

Prions template conformational conversion of other molecules of the same protein

Prions are disseminated to daughter cells during cell division

Prions are formed through an oligomeric nucleus, and the elongating polymer is severed by protein remodeling factors

from Halfmann and Lindquist, Science 330, 629 (2010)

Stress Accelerates Prion Appearance

from Halfmann and Lindquist, Science 330, 629 (2010)

Prion-free cells are adapted to environment 1, but poorly adapted to environment 2

Prion formation and disappearance provide fitness advantages in different environments

Abrupt changes have consequences for protein folding

Prions connect environmental conditions to acquisition and inheritance of new traits

Co-suppression

PcG complexes interact in trans

Increase in gene copy number results in decreased expression

Dependent on PcG genes

from Pirrotta, Cell 93, 333 (1998)

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

Imprinted Expression of the H19 and Igf2 Genes

from Reik and Murrell, Nature 405, 408 (2000)

Maternal – H19 expression

Paternal – Igf2 expression

Imprinting is Regulated by a Methylation-sensitive Boundary

from Reik and Murrell, Nature 405, 408 (2000)

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 Ferguson-Smith and Surani, Science 293, 1086 (2001)

Imprinting of the PWS-AS Locus

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

from Straub and Becker, Nature Rev.Genet. 8, 47 (2007)

Dosage Compensation Mechanisms

Genomes compensate for different numbers of sex chromosomes by adjusting gene expression levels

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

Model for the Initiation of X Inactivation

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 22-7

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

Stepwise Progression of X Inactivation in Differentiating ES Cells

from Brockdorff, Trends Genet. 18, 352 (2002)

One X chromosome is converted to facultative heterochromatin

Xist transcription off the inactive X initiates chromatin modification events

X inactivation is maintained epigenetically

Calico Cats

One of the genes controlling fur color is on the X chromosome

B – orangeb - black

Random X inactivation early in embryonic development leads to patchworks of skin cells expressing each allele

Female mammals are genetic mosaics

The Dosage Compensation Complex in Drosophila

from Gilfillan et al., FEBS Lett. 567, 8 (2004)

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

H4K16 acetylation impedes formation of condensed chromatin structure

from Straub and Becker, Nature Rev.Genet. 8, 47 (2007)

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