Heterochromatin: Dark, Silent, and Propagating Structure

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. 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., Nature410, 120 (2001) H1 recruits Su(var)3-9 to heterochromatin resulting in transposon silencing

5. 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

6. Position Effect Variegation from Brummelkamp and van Steensel, Science348, 1433 (2015) Expression levels of transgenes is dependent on integration site Integration into heterochromatin results in repression H3K9me recruits HUSH complex SETDB1 methylates H3K9 to enable spread of heterochromatin

7. 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)

8. 3D Organization of the Genome Contributes to Controlling Gene Expression CTCF mediates long-range interactions between genomic sequences Genes within topologically associating domains (TADs) exhibit correlated expression from Ong and Corces, Nature Rev.Genet. 15, 234 (2014)

9. Assays for Barrier Insulator Activity Barrier insulators protect transgene from position-effect silencing Functional borders may require several different mechanisms including CTCF binding The function of insulators is to maintain higher-order folding and establish TADs From Phillips-Cremins and Corces, Mol.Cell 50, 461 (2013)

10. Isocitrate Dehydrogenase Mutation are Correlated with Cancer from Grimmer and Costello, Nature529, 34 (2016) Mutant IDH converts isocitrate into a metabolite that inhibits TET Reduced CTCF binding alters chromatin conformation A potent enhancer interacts with an oncogene promoter Modulating 3D chromatin structure may be widespread in cancer

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. 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, Nature477, 169 (2011)

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

15. 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

16. Epigenetic Modifications Within an Arabidopsis Chromosome Heterochromatin correlates with epigenetic marks from Zhang, Science320, 489 (2008)

17. DNA Methylation Methylation at CpG residues correlates with gene repression 5meC is involved in stable epigenetic repression Sites of methylation Inactive X Imprinted loci Transposon-derived sequences CpG islands are CpG-rich regions usually found at promoters Methylation patterns are reproduced at each round of cell division

18. 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

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

20. 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

21. 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)

22. 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., BioEssays32, 669 (2010)

23. DNA is Demethylated by TET Proteins 5mC is oxidated iteratively by TET 5hmC is reverted to unmodified C by passive dilution during DNA replication Oxidative products are excised by thymine DNA glycosylase and repaired by BER from Kohli and Zhang, Nature502, 472 (2013)

24. Rett Syndrome Onset of symptoms in humans is 6-12 months of age Rett syndrome is a severe neurological disorder Abnormal head growth Decrease in speech function Breathing disturbances Repetitive hand movements Caused by mutation in the MeCP2 gene on the X chromosome

25. MeCP2 Function and Rett Syndrome from Lyst and Bird, Nature Rev.Genet. 16, 261 (2015) MeCP2 binds methylated DNA, but also binds to unmethylated DNA via regions outside the MBD MeCP2 compacts chromatin structure MeCP2 recruits a corepressor complex that contains a histone deacetylase

26. MeCP2 Regulates Gene Expression in Response to Neural Activity MeCP2 binds methylated DNA and silences target genes such as BDNF and corticotropin-releasing hormone Neural activity triggers MeCP2 phosphorylation and target gene activation from Bienvenu and Chelly, Nature Rev.Genet.7, 415 (2006) Hippocampal neurons grow dendrites with fewer branches when MeCP2 is blocked Increase in mCH sequences after birth is enriched in genes with neuronal functions from Miller, Science314, 1356 (2006)

27. Many MeCP2-Repressed Genes Encode Proteins That Modulate Neuronal Physiology MeCP2 binds mCA Density of mCA is higher in long genes Frequency of mCA increases with age Long genes are selectively expressed in the brain Length- and mCA-dependent increase in gene expression in MeCP2 mutants from Gabel et al., Nature522, 89 (2015) from Luo and Ecker, Science348, 1094 (2015)