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 Modified histones recruit HP1 HP1 recruits histone modifying enzymes to facilitate heterochromatin spread Boundary elements prevent further heterochromatin spread

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 from Bannister et al., Nature 410, 120 (2001) H1 recruits Su(var)3-9 to heterochromatin resulting in transposon silencing

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)

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)

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 H3K9 methylation by Clr4 Methylated H3K9 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)

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)

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 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)

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 Stable block interrupts propagation of heterochromatin Active barrier recruits a complex containing chromatin remodeling activity from Donze and Kamakaka, BioEssays 24, 344 (2002)

BRCA1 Modifies Pericentric Heterochromatin BRCA1 promotes enrichment of Ub-H2A in pericentric heterochromatin Loss of BRCA1 triggers transcription of satellite-DNA in pericentric heterochromatin Satellite-DNA transcription is sufficient to induce genome instability after loss of BRCA1 from Venkitaraman, Nature 477, 169 (2011)

Epigenetics Heritable changes in gene function that cannot be explained by changes in gene sequences DNA methylation Histone variants and modifications Nucleosome positioning

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

Epigenetic Modifications Within an Arabidopsis Chromosome Heterochromatin correlates with epigenetic marks from Zhang, Science 320, 489 (2008)

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 from Portela and Esteller, Nature Biotechnol. 28, 1057 (2010) More than half of human promoters contain CpG islands Promoters are usually unmethylated 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

Methylated CpG Islands Inhibit Transcription from Portela and Esteller, Nature Biotechnol. 28, 1057 (2010) More than half of human promoters contain CpG islands Promoters are usually unmethylated 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

Methylation of Repetitive Sequences Stabilize Chromosomes from Portela and Esteller, Nature Biotechnol. 28, 1057 (2010) Unmethylated repetitive sequences cause reactivation of endoparasitic sequences

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 from Law and Jacobsen, Nature Rev.Genet. 11, 204 (2010) siRNA-IGN duplex is formed and recruits DRM2

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 from Law and Jacobsen, Nature Rev.Genet. 11, 204 (2010) Tetramer oligomerizes and results in 10 bp pattern of methylation on the same strand

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)

Propagation of DNA Methylation State Newly synthesized methylated DNA is hemimethylated NP95 binds hemimethylated DNA DNMT1 is a maintenance methyltransferase and binds PCNA NP95 links DNMT1 to hemimethylated DNA from Richly et al., BioEssays 32, 669 (2010)

Mechanisms for Repression Mediated by MBD Proteins from Wade, BioEssays 23, 1131 (2001)

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)