The involvement of histone modifications in higher-order chromatin assembly has been highlighted by our studies in S. pombe showing that several factors identified in genetic screen for mutants defective in heterochromatic silencing (such as such as Clr3, Clr4 and Clr6) are involved in modifications of histone tails. Among these, Clr4 belongs to a highly conserved Suv39 family of histone methyltransferases, which specifically methylate histone H3 at lysine 9 (H3-K9) across heterochromatic domains associated with repetitive DNA elements. Biochemical analysis has shown that Clr4 is a component of multisubunit complex containing a cullin family protein Cul4 that serves as scaffold to assemble ubiquitin ligases, and a WD protein Rik1 which mediates recruitment of Clr4 activity to the target repeat loci via a RNA polymerase II transcription coupled process. Clr3 and Clr6 are histone decaetylases with strong homologies to class II and class I HDACs from humans. We have shown that Clr6 exists in at least two distinct core complexes. One of these complexes (Clr6-C1) predominantly targets gene promoters and is responsible for regulation of gene expression through local deacetylation of histones. The second Clr6 complex (Clr6-CII) that targets transcribed chromosomal regions and centromeric loci is responsible for global deacetylation of histones. Our analyses suggest that defects in Clr6-CII abrogate global protective functions of chromatin such as suppression of antisense transcripts, strand-specific repression of heterochromatic repeats and protection of DNA from damage by genotoxic agents. We have also performed biochemical characterization of Clr3. Clr3 exists in a multienzyme effector complex termed SHREC that in addition to histone decaetylase activity associated with Clr3 contains a Snf2 family chromatin remodeling factor Mit1. We have shown that SHREC is targeted across all major heterochromatic domains and its activities are essential for proper positioning of nucleosomes to assemble higher-order chromatin structures, critical for heterochromatin functions. We are continuing to investigate the functions of these and other histone modifying activities. Given that histone modifiers are conserved among species and control fundamental chromosomal processes including stable maintenance of gene expression patterns during development and maintenance of genomic integrity, their deeper understanding is important for the development of effective therapeutic measures for treatment of cancer and other human diseases. Heterochromatin nucleated at specific sites spread in a manner that depends upon the activities of histone decaetylases, heterochromatin proteins and the ability of Clr4 to both methylate H3-K9 as well as bind to methylated H3 tail via its chromodomain. Moreover, methylation of H3-K9 is essential for recruitment of HP1 proteins such as Swi6, Chp2 and Chp1. Our research has unraveled a new theme wherein HP1 proteins bound to methylated H3-K9 provide a dynamic platform for factors involved in many cellular processes, including proteins involved in cell-type switching and proper segregation of chromosomes. Chp1, a component of the RITS complex tethers RNAi machinery to heterochromatic loci, facilitating post-transcriptional silencing of repeats in cis. However, the exact functions of Chp2 and Swi6 in heterochromatin assembly and their associations with other factors were poorly understood. We recently showed that Swi6 and Chp2 associate with Clr6 and SHREC histone deacetylase complexes, which are critical for transcriptional silencing of the heterochromatic centromeric repeats. This work further revealed that Swi6 and Chp2 proteins and their associated HDAC complexes have overlapping functions in limiting RNA polymerase II occupancy across pericentromeric heterochromatin domains. Interestingly, purified Swi6 fraction also contains factors involved in a variety of chromosomal processes such as chromatin remodeling and DNA replication. In addition, Swi6 co-purifies a cohesin loading factor essential for sister chromatid cohesion, and with centromere-specific histone H3 variant CENP-A, which is incorporated into chromatin in a heterochromatin-dependent manner. These analyses suggest that HP1 proteins associate with a variety of factors including histone-modifying factors essential for the assembly of repressive chromatin. Identifications of HP1 associated factors and their role in chromatin assembly may help us understand the causes of breast cancer associated with altered HP1 expression. Although HP1 proteins are critical for the preferential recruitment of histone deacetylases to repeat elements within heterochromatin domains, alternative mechanisms exist to target these activities to repeats dispersed across the genome. Specifically, we have uncovered a novel genome surveillance mechanism for retrotransposons by a family of transposase-derived CENP-B homologs. We found that CENP-Bs localize at and recruit histone deacetylases to silence retrotransposons. This mechanism also represses retrotransposon relics scattered throughout the S. pombe genome. CENP-B-mediated surveillance is proactive, capable of preventing an extinct retrotransposon from reentering the host genome. These results reveal a likely ancient retrotransposon surveillance pathway and suggest that eukaryotic cells have a toolkit of repressor activities that are either targeted across large domains via HP1 proteins or in a site-specific manner by CENP-B and other DNA binding factors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011208-05
Application #
8763400
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2013
Total Cost
$1,488,146
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Mizuguchi, Takeshi; Barrowman, Jemima; Grewal, Shiv I S (2015) Chromosome domain architecture and dynamic organization of the fission yeast genome. FEBS Lett 589:2975-86
Folco, H Diego; Campbell, Christopher S; May, Karen M et al. (2015) The CENP-A N-tail confers epigenetic stability to centromeres via the CENP-T branch of the CCAN in fission yeast. Curr Biol 25:348-56
Mizuguchi, Takeshi; Fudenberg, Geoffrey; Mehta, Sameet et al. (2014) Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe. Nature 516:432-5
Anver, Shajahan; Roguev, Assen; Zofall, Martin et al. (2014) Yeast X-chromosome-associated protein 5 (Xap5) functions with H2A.Z to suppress aberrant transcripts. EMBO Rep 15:894-902
Aygun, Ozan; Mehta, Sameet; Grewal, Shiv I S (2013) HDAC-mediated suppression of histone turnover promotes epigenetic stability of heterochromatin. Nat Struct Mol Biol :
Shim, Young Sam; Choi, Yoonjung; Kang, Keunsoo et al. (2012) Hrp3 controls nucleosome positioning to suppress non-coding transcription in eu- and heterochromatin. EMBO J 31:4375-87
Lorenz, David R; Mikheyeva, Irina V; Johansen, Peter et al. (2012) CENP-B cooperates with Set1 in bidirectional transcriptional silencing and genome organization of retrotransposons. Mol Cell Biol 32:4215-25
Zhang, Ke; Fischer, Tamas; Porter, Rebecca L et al. (2011) Clr4/Suv39 and RNA quality control factors cooperate to trigger RNAi and suppress antisense RNA. Science 331:1624-7
Matsuda, Emiko; Sugioka-Sugiyama, Rie; Mizuguchi, Takeshi et al. (2011) A homolog of male sex-determining factor SRY cooperates with a transposon-derived CENP-B protein to control sex-specific directed recombination. Proc Natl Acad Sci U S A 108:18754-9
Ni, Ting; Tu, Kang; Wang, Zhong et al. (2010) The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe. PLoS One 5:e15271

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