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, Science301, 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, Cell93, 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, Cell93, 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, BioEssays24, 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, Science320, 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, BioEssays23, 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, Science314, 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, Science318, 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, Cell93, 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, Science293, 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)