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, 6 th ed. Fig 6-33

Histone Modifications Associated with Heterochromatin and Euchromatin from Lodish et al., Molecular Cell Biology, 6 th 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 heterochromatin from Schramke and Allshire, Science 301, 1069 (2003)

Formation of Telomeric Heterochromatin from Grunstein, Cell 93, 325 (1998) RAP1 binds to C 1-3 A 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, 6 th 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)

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 DNMT1 to 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, 5 th ed. Fig 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, 5 th 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, 6 th ed. Fig 22-7

Model for the Initiation of X Inactivation from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 22-7 The Xic in female cells colocalize prior to X inactivation Low expression of Tsix from X i leads to Xist transcription Xist RNA coats the X i in cis The chromatin structure of X i 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 – orange b - 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