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A paucity of heterochromatin at functional human neocentromeres
Alicia Alonso, Dan Hasson, Fanny Cheung, Peter E Warburton
Epigenetics & Chromatin , 2010, DOI: 10.1186/1756-8935-3-6
Abstract: High-resolution chromatin immunoprecipitation (ChIP) on CHIP (microarray) analysis of three independent neocentromeres from chromosome 13q revealed that each neocentromere contained ~100 kb of centromere protein (CENP)-A in a two-domain organization. Additional CENP-A domains were observed in the vicinity of neocentromeres, coinciding with CpG islands at the 5' end of genes. Analysis of histone H3 dimethylated at lysine 4 (H3K4me2) revealed small domains at each neocentromere. However, these domains of H3K4me2 were also found in the equivalent non-neocentric chromosomes. A surprisingly minimal (~15 kb) heterochromatin domain was observed at one of the neocentromeres, which formed in an unusual transposon-free region distal to the CENP-A domains. Another neocentromere showed a distinct absence of nearby significant domains of heterochromatin. A subtle defect in centromere cohesion detected at these neocentromeres may be due to the paucity of heterochromatin domains.This high-resolution mapping suggests that H3K4me2 does not seem sufficiently abundant to play a structural role at neocentromeres, as proposed for endogenous centromeres. Large domains of heterochromatin also do not appear necessary for centromere function. Thus, this study provides important insight into the structural requirements of human centromere function.The centromere is the chromosomal locus responsible for the proper segregation of replicated sister chromatids to daughter cells during cell division. In all eukaryotes, the centromere is characterized by a unique chromatin structure that contains a centromere-specific histone 3 variant, called centromere protein (CENP)-A in mammals [1,2]. The kinetochore, a large multiprotein complex, is built onto this CENP-A chromatin and mediates microtubule attachment during mitosis and meiosis [3]. The CENP-A domain is flanked by heterochromatin, characterized by histone H3 methylated at lysine 9 (H3K9me), which may be important for centromeric chromatid cohe
Neocentromeres Form Efficiently at Multiple Possible Loci in Candida albicans  [PDF]
Carrie Ketel equal contributor,Helen S. W. Wang equal contributor,Mark McClellan,Kelly Bouchonville,Anna Selmecki,Tamar Lahav,Maryam Gerami-Nejad,Judith Berman
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000400
Abstract: Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-ACse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.
LINE Retrotransposon RNA Is an Essential Structural and Functional Epigenetic Component of a Core Neocentromeric Chromatin  [PDF]
Anderly C. Chueh,Emma L. Northrop,Kate H. Brettingham-Moore,K. H. Andy Choo ,Lee H. Wong
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000354
Abstract: We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10) containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A–binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A–binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s) across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A–binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A–associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.
Plasticity of Fission Yeast CENP-A Chromatin Driven by Relative Levels of Histone H3 and H4  [PDF]
Araceli G Castillo equal contributor,Barbara G Mellone equal contributor,Janet F Partridge,William Richardson,Georgina L Hamilton,Robin C Allshire ,Alison L Pidoux
PLOS Genetics , 2007, DOI: 10.1371/journal.pgen.0030121
Abstract: The histone H3 variant CENP-A assembles into chromatin exclusively at centromeres. The process of CENP-A chromatin assembly is epigenetically regulated. Fission yeast centromeres are composed of a central kinetochore domain on which CENP-A chromatin is assembled, and this is flanked by heterochromatin. Marker genes are silenced when placed within kinetochore or heterochromatin domains. It is not known if fission yeast CENP-ACnp1 chromatin is confined to specific sequences or whether histone H3 is actively excluded. Here, we show that fission yeast CENP-ACnp1 can assemble on noncentromeric DNA when it is inserted within the central kinetochore domain, suggesting that in fission yeast CENP-ACnp1 chromatin assembly is driven by the context of a sequence rather than the underlying DNA sequence itself. Silencing in the central domain is correlated with the amount of CENP-ACnp1 associated with the marker gene and is also affected by the relative level of histone H3. Our analyses indicate that kinetochore integrity is dependent on maintaining the normal ratio of H3 and H4. Excess H3 competes with CENP-ACnp1 for assembly into central domain chromatin, resulting in less CENP-ACnp1 and other kinetochore proteins at centromeres causing defective kinetochore function, which is manifest as aberrant mitotic chromosome segregation. Alterations in the levels of H3 relative to H4 and CENP-ACnp1 influence the extent of DNA at centromeres that is packaged in CENP-ACnp1 chromatin and the composition of this chromatin. Thus, CENP-ACnp1 chromatin assembly in fission yeast exhibits plasticity with respect to the underlying sequences and is sensitive to the levels of CENP-ACnp1 and other core histones.
Factors That Promote H3 Chromatin Integrity during Transcription Prevent Promiscuous Deposition of CENP-ACnp1 in Fission Yeast  [PDF]
Eun Shik Choi,Annelie Str?lfors,Sandra Catania,Araceli G. Castillo,J. Peter Svensson,Alison L. Pidoux,Karl Ekwall,Robin C. Allshire
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002985
Abstract: Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-ACnp1 deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-ACnp1 incorporation at non-centromeric sites. FACT has little or no effect on CENP-ACnp1 assembly at endogenous centromeres where CENP-ACnp1 is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-ACnp1 at specific loci, including subtelomeric regions, where CENP-ACnp1 is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-ACnp1 chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-ACnp1 to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-ACnp1 chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification.
Properly discontinuous actions on bounded domains  [PDF]
Bo-Yong Chen
Mathematics , 2008,
Abstract: We give sufficient conditions for the quotient of a free, properly discontinuous action on a bounded domain of holomorphy to be a Stein manifold in terms of Poincar\'e series or limit sets for orbits. An immediate consequence is that the quotient of any cyclic, free, properly discontinuous action on the unit ball or the bidisc is Stein.
The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C  [PDF]
Elaine M. Dunleavy equal contributor,Nicole L. Beier equal contributor,Walter Gorgescu,Jonathan Tang,Sylvain V. Costes,Gary H. Karpen
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1001460
Abstract: CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.
Multiscale Identification of Topological Domains in Chromatin  [PDF]
Darya Filippova,Rob Patro,Geet Duggal,Carl Kingsford
Quantitative Biology , 2013,
Abstract: Recent chromosome conformation capture experiments have led to the discovery of dense, contiguous, megabase-sized topological domains that are similar across cell types and conserved across species. These domains are strongly correlated with a number of chromatin markers and have since been included in a number of analyses. However, functionally-relevant domains may exist at multiple length scales. We introduce a new and efficient algorithm that is able to capture persistent domains across various resolutions by adjusting a single scale parameter. The identified novel domains are substantially different from domains reported previously and are highly enriched for insulating factor CTCF binding and histone modfications at the boundaries.
A Coordinated Interdependent Protein Circuitry Stabilizes the Kinetochore Ensemble to Protect CENP-A in the Human Pathogenic Yeast Candida albicans  [PDF]
Jitendra Thakur,Kaustuv Sanyal
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002661
Abstract: Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3–5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.
Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis  [PDF]
Barbara G. Mellone ,Kathryn J. Grive equal contributor,Vladimir Shteyn equal contributor,Sarion R. Bowers,Isaac Oderberg,Gary H. Karpen
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002068
Abstract: Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.
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