The higher-order assembly of chromatin imposes structural organization on the genetic information of eukaryotes and is thought to be largely determined by posttranslational modification of histone tails. Here, we study a 20-kilobase silent domain at the mating-type region of fission yeast as a model for heterochromatin formation. We find that, although histone H3 methylated at lysine 9 (H3 Lys9) directly recruits heterochromatin protein Swi6/HP1, the critical determinant for H3 Lys9 methylation to spread in cis and to be inherited through mitosis and meiosis is Swi6 itself. We demonstrate that a centromere-homologous repeat (cenH) present at the silent mating-type region is sufficient for heterochromatin formation at an ectopic site, and that its repressive capacity is mediated by components of the RNA interference (RNAi) machinery. Moreover, cenH and the RNAi machinery cooperate to nucleate heterochromatin assembly at the endogenous mat locus but are dispensable for its subsequent inheritance. This work defines sequential requirements for the initiation and propagation of regional heterochromatic domains.
Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.
The heterochromatin domain at the mat locus of Schizosaccharomyces pombe is bounded by the IR-L and IR-R barriers. A genetic screen for mutations that promote silencing beyond IR-L revealed a novel gene named epe1, encoding a conserved nuclear protein with a jmjC domain. Disruption of epe1 promotes continuous spreading of heterochromatin-associated histone modifications and Swi6 binding to chromatin across heterochromatic barriers. It also enhances position effect variegation at heterochromatic domains, suppresses mutations in silencing genes, and stabilizes the repressed epigenetic state at the mat locus. However, it does not enhance silencing establishment. Our analysis suggests that the jmjC domain is essential for Epe1 activity and that Epe1 counteracts transcriptional silencing by negatively affecting heterochromatin stability. Consistent with this proposition, the meiotic stability of established heterochromatin beyond IR-L is diminished by Epe1 activity, and overexpression of Epe1 disrupts heterochromatin through acetylation of H3-K9 and H3-K14 and methylation of H3-K4. Furthermore, overexpression of Epe1 elevates the rate of chromosome loss. We propose that Epe1 helps control chromatin organization by down-regulating the stability of epigenetic marks that govern heterochromatization.Epigenetic control of gene expression by clonally inherited higher-order chromatin structures accounts for diverse biological phenomena, such as dosage compensation, imprinting, and position effect variegation (PEV) (26,44,45). In addition to transcription regulation, epigenetic effects have an important impact on cellular differentiation, accurate chromosome segregation, recombination, neocentromere maintenance, and mating-type switching in yeast (reviewed in references 1, 18, 24, 25, 28, 37, and 47). Molecular models that explain chromosomal inheritance of epigenetic states assume a long-range memory mechanism that marks imprinted chromosomal domains by self-templating higher-order chromatin structures and covalent modification of DNA (20,46). Evidence for chromatin modifications in imprinted domains has been obtained through experiments that demonstrate the association of silent chromosomal regions with modified histones and chromatinassociated nonhistone proteins like Saccharomyces cerevisiae SIR3, Drosophila HP1, and Schizosaccharomyces pombe Swi6 (15,22,33,36).The mat locus of S. pombe shares an extended sequence homology with the centromeric outer repeats (cenH) and provides a useful paradigm for the highly conserved process of heterochromatin assembly and inheritance (17,18,25). Constitutive heterochromatin extends along the 15-kb mat2-K-mat3 interval that is bound on its centromere-proximal end by the REII protosilencer (Fig. 1A) (7,21,33,41). Silencing diminishes gradually at the L region as the distance from REII toward the IR-L heterochromatin barrier increases (6). IR-L, like its IR-R homologous counterpart at the centromere-distal end of the heterochromatic domain, is a distinct transition point for Swi6 associa...
Members of the lysine (K)-specific demethylase 4 (KDM4) A-D family of histone demethylases are dysregulated in several types of cancer. Here, we reveal a previously unrecognized role of KDM4D in the DNA damage response (DDR). We show that the C-terminal region of KDM4D mediates its rapid recruitment to DNA damage sites. Interestingly, this recruitment is independent of the DDR sensor ataxia telangiectasia mutated (ATM), but dependent on poly (ADP-ribose) polymerase 1 (PARP1), which ADP ribosylates KDM4D after damage. We demonstrate that KDM4D is required for efficient phosphorylation of a subset of ATM substrates. We note that KDM4D depletion impairs the DNA damage-induced association of ATM with chromatin, explaining its effect on ATM substrate phosphorylation. Consistent with an upstream role in DDR, KDM4D knockdown disrupts the damage-induced recombinase Rad51 and tumor protein P53 binding protein foci formation. Consequently, the integrity of homology-directed repair and nonhomologous end joining of DNA breaks is impaired in KDM4D-deficient cells. Altogether, our findings implicate KDM4D in DDR, furthering the links between the cancer-relevant networks of epigenetic regulation and genome stability.histone demethylation | chromosome instability | PARylation
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