Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Telomeric repeats facilitate CENP-A incorporation via telomere binding proteins

ABSTRACT: The histone H3 variant, CENP-ACnp1, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-ACnp1 deposition we investigated whether certain locations are favoured when additional CENP-ACnp1 is present in fission yeast cells. Our analyses show that additional CENP-ACnp1 accumulates within and close to heterochromatic centromeric outer repeats, and over regions adjacent to rDNA and telomeres. The use of minichromosome derivatives with unique DNA sequences internal to chromosome ends shows that telomeres are sufficient to direct CENP-ACnp1 deposition. However, chromosome ends are not required as CENP-ACnp1 deposition also occurs at telomere repeats inserted at an internal locus and correlates with the presence of H3K9 methylation near these repeats. The Ccq1 protein, which is known to bind telomere repeats and recruit telomerase, was found to be required to induce H3K9 methylation and thus promote the incorporation of CENP-A near telomere repeats. These analyses demonstrate that at non-centromeric chromosomal locations the presence of heterochromatin influences the sites at which CENP-A is incorporated into chromatin and thus, potentially the location of centromeres. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from mutants and wild type control cells in biological duplicates normalized to input DNA from each strain.

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Karl Ekwall

PROVIDER: E-GEOD-46427 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins.

Castillo Araceli G AG Pidoux Alison L AL Catania Sandra S Durand-Dubief Mickaël M Choi Eun Shik ES Hamilton Georgina G Ekwall Karl K Allshire Robin C RC

PloS one 20130731 7

The histone H3 variant, CENP-A, is normally assembled upon canonical centromeric sequences, but there is no apparent obligate coupling of sequence and assembly, suggesting that centromere location can be epigenetically determined. To explore the tolerances and constraints on CENP-A deposition we investigated whether certain locations are favoured when additional CENP-A(Cnp1) is present in fission yeast cells. Our analyses show that additional CENP-A(Cnp1) accumulates within and close to heteroch ...[more]

PMID: 23936074

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Project description: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-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) 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-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) 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-A(Cnp1) 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-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification. In total, 24 samples: 22 ChIP DNA files (10 different conditions), 2 Input files.

Project description:Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays we show that Top3 unlike Top1 and Top2 is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology which in turn affects the dynamics of CENP-ACnp1 nucleosomes. For transcription: Total RNA from top3-105 mutant and WT control cells after 8 hours at 36C in biological duplicates. For Top3-myc chromatin immunoprecipitation: DNA immunoprecipitated with mouse anti-Myc using chromatin extracts from cells expressing Top3-Myc from the endogenous locus at 30C in biological duplicates normalized to input DNA from wild type cells at 30C in biological duplicates. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from top3-105 mutant and wild type control cells after 8 hours at 36C in in biological duplicates normalized to input DNA from each strain.

Project description:Functional centromeres are essential for proper cell division. Centromeres are established largely by epigenetic processes resulting in incorporation of the histone H3 variant CENP-A. Here, we demonstrate the direct involvement of H2B monoubiquitination, mediated by RNF20 in humans or Brl1 in Schizosaccharomyces pombe, in centromeric chromatin maintenance. Monoubiquinated H2B (H2Bub1) is needed for this maintenance, promoting noncoding transcription, centromere integrity and accurate chromosomal segregation. A transient pulse of centromeric H2Bub1 leads to RNA polymerase IIM-bM-^@M-^Smediated transcription of the centromereM-bM-^@M-^Ys central domain, coupled to decreased H3 stability. H2Bub1-deficient cells have centromere cores that, despite their intact centromeric heterochromatin barriers, exhibit characteristics of heterochromatin, such as silencing histone modifications, reduced nucleosome turnover and reduced levels of transcription. In the H2Bub1-deficient cells, centromere functionality is hampered, thus resulting in unequal chromosome segregation. Therefore, centromeric H2Bub1 is essential for maintaining active centromeric chromatin. ChIP: In total 17 samples; 15 ChIP DNA files (7 different conditions) 2 Input files. Cnp1_WT_input.bar (antibody against Cnp1 in strains Hu303, Hu29 (WT) (Cnp1_Hu303.CEL, AS_10.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); Cnp1_Hu2640_WT.bar (antibody against Cnp1 in strain Hu2640 (htb1K119R) (Cnp1_Hu2640_A.CEL, Cnp1_Hu2640_B.CEL) vs strains Hu303, Hu29 (WT) (Cnp1_Hu303.CEL, AS_10.CEL)); Cnp1_Hu2640_input.bar (antibody against Cnp1 in strain Hu2640 (htb1K119R) (Cnp1_Hu2640_A.CEL, Cnp1_Hu2640_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H2Bub1_Hu303_input.bar (antibody against H2Bub1 in strain Hu303 (WT) (H2Bub1_Hu303_A.CEL, H2Bub1_Hu303_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3_WT_input.bar (antibody against H3 in strains Hu303, Hu29 (WT) (H3_Hu303.CEL, As_12.CEL) vs input (JW_1.CEL, JW_2_2.CEL); H3_Hu2640_WT.bar (antibody against H3 in strain Hu2640 (htb1K119R) (H3_Hu2640_A.CEL, H3_Hu2640_B.CEL, H3_Hu2640_C.CEL) vs strains Hu303, Hu29 (WT) (H3_Hu303.CEL, AS_12.CEL)); H3_Hu2640_input.bar (antibody against H3 in strain Hu2640 (htb1K119R) (H3_Hu2640_A.CEL, H3_Hu2640_B.CEL, H3_Hu2640_C.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3K9me2_Hu303_input.bar (antibody against H3K9me2 in strains Hu303, Hu29 (WT) (H3K9me2_Hu303.CEL, AS_6.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3k9me2_Hu2640_Hu303.bar (antibody against H3K9me2 in strain Hu2640 (htb1K119R) (H3K9me2_Hu2640_A.CEL, H3K9me2_Hu2640_B.CEL) vs strain Hu303, Hu29 (WT) (H3K9me2_Hu303.CEL, AS_6.CEL)); H3K9me2_Hu2640_input.bar (antibody against H3K9me2 in strain Hu2640 (htb1K119R) (H3K9me2_Hu2640_A.CEL, H3K9me2_Hu2640_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)). RNA: 4 RNA samples: 2 replicates of WT RNA (RNA_Hu303_A.CEL, RNA_Hu303_B.CEL) and 2 replicates of htb1-K119R RNA (RNA_Hu2640_A.CEL, RNA_Hu2640_B.CEL).

Project description:Heterochromatin plays a key role in gene repression, maintaining genome integrity and chromosome segregation. Fission yeast, Schizosaccharomyces pombe, utilizes conserved components to direct heterochromatin formation using siRNA generated by RNA interference (RNAi) to guide a histone H3 lysine 9 methyltransferase to cognate chromatin. To identify compounds that inhibit heterochromatin formation, an in vivo phenotypic screen for loss of silencing was performed. A tester strain harbouring a silent dominant selectable kanMX reporter gene within fission yeast centromeric heterochromatin was used to screen a diverse library of chemicals. HMS-I1 and HMS-I2 were identified as compounds that reproducibly increased G418 resistance due to loss of kanMX silencing, and decreased the level of repressive H3K9 methylation on centromeric repeats. The pattern of changes induced by HMS-I1 and HMS-I2 were consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic interactions and expression profiling indicated that both HMS-I1 and HMS-I2 affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). Exposure to HMS-I1 was also found to alleviate silencing of reporter genes in an Arabidopsis transgenic plant line and a mouse cell line. HMS-I2 also disrupted reporter gene silencing in Arabidopsis. In vitro assays indicate that HMS-I1 impairs the activity of human HDAC6 and HDAC10. As HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities they represent potentially novel and valuable reagents for experimental and therapeutic purposes. Our findings highlight the use of in vivo chemical screening conducted in fission yeast to identify compounds that disrupt heterochromatin across plant, fungi and animal kingdoms. 16 RNA samples: 2 replicates of WT untreated (vehicle DMSO) (KE1-A, KE2-A), HMS-I1 treated (KE1-B, KE2-B), HMS-I2 treated (KE1-C, KE2-C), and 2 additional replicates of WT (KE05_wt_1, KE10_wt_2), 4 replicates of clr2M-bM-^HM-^F (KE01_2118_1, KE06_2118_2, KE03_0080_1, KE08_0080_2) and mit1M-bM-^HM-^F (KE02_1295_1, KE07_1295_2, KE04_1278_1, KE09_1278_2).

Dataset Information

Telomeric repeats facilitate CENP-A incorporation via telomere binding proteins

Publications

Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins.

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