Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

CENP-B cooperates with Set1 in bidirectional transcriptional silencing and genome organization of retrotransposons

ABSTRACT: Expression profiling of mutant cells compared to wild-type cells. Expression profiling of S. pombe CENP-B homolog cbp1M-bM-^HM-^F (abp1/SPBC1105.04c) and histone deacetylase double mutant clr3M-bM-^HM-^FM-BM- clr6-1 (SPBC800.03 SPBC36.05c) cells. Wildtype and mutant cDNA was reverse transcribed from total RNA using the Invitrogen SuperscriptIII Indirect labeling kit and the supplied anchored oligo-dT according to the manufacturer's instructions. cDNA from mutant and wildtype samples were labeled with Alexa Fluor 555 and 647 dyes, respectively. Biological duplicates were performed for each mutant and wildtype sample. An Agilent 44K in situ 60mer DNA microarray that tiles the entire Schizosaccharomyces pombe genome every 300 bp alternately on both strands was used to probe expression of transcripts in CENP-B homolog cbp1M-bM-^HM-^F (abp1/SPBC1105.04c) and histone deacetylase double mutants clr3M-bM-^HM-^F clr6-1 (SPBC800.03 SPBC36.05c) cells versus wildtype.

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Hugh Cam

PROVIDER: E-GEOD-39404 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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CENP-B cooperates with Set1 in bidirectional transcriptional silencing and genome organization of retrotransposons.

Lorenz David R DR Mikheyeva Irina V IV Johansen Peter P Meyer Lauren L Berg Anastasia A Grewal Shiv I S SI Cam Hugh P HP

Molecular and cellular biology 20120820 20

Regulation of transposable elements (TEs) is critical to the integrity of the host genome. The fission yeast Schizosaccharomyces pombe homologs of mammalian CENP-B perform a host genome surveillance role by controlling Tf2 long terminal repeat (LTR) retrotransposons. However, the mechanisms by which CENP-Bs effect their functions are ill defined. Here, we show that the multifaceted roles of Abp1, the prominent member of fission yeast CENP-Bs, are mediated in part via recognition of a 10-bp AT-ri ...[more]

PMID: 22907751

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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:Proper chromatin organization is essential for defining transcription units and maintaining genomic integrity in eukaryotes. Mutations affecting the chromatin structure can lead to increased cryptic transcription and genomic instability. In this study we found that deletion of the Schizosaccharomyces pombe Chd1-type chromatin remodelers, hrp1 and hrp3, causes strong, genome-wide accumulation of antisense transcripts, while the amount of coding mRNA transcripts is mostly unaffected. Nucleosome mapping revealed a specific role for Chd1-remodelers in the positioning of nucleosomes in gene coding regions. While the arrangement of nucleosomes in promoter regions was similar to WT, nucleosome organization within coding regions was remarkably irregular in hrp1M-bM-^HM-^Fhrp3M-bM-^HM-^F strain. We extended our analysis to other mutations associated with enhanced cryptic transcription activity, such as set2M-bM-^HM-^F, alp13M-bM-^HM-^F, and FACT complex subunit pob3M-bM-^HM-^F. While nucleosomes were severely depleted in the pob3M-bM-^HM-^F strain, nucleosome positioning was less affected. In sharp contrast, nucleosome organization in the alp13M-bM-^HM-^F and set2M-bM-^HM-^F strains was indistinguishable from WT. These data indicate multiple mechanisms in the repression of cryptic promoter activity in eukaryotic cells. Genome-wide profiling of H3K9/K14 acetylation Genome-wide expression analysis of either Alp13-, Set2-, Hrp3 or Hrp1 and Hrp3-deficient cells Genome-wide expression analysis of either Hrp1, Hrp3, or Hrp1 and Hrp3-deficient cells Nucleosome mapping experiments ChIP for the detection of the genome-wide acetylation profile of H3K9/K14 was performed for a wildtype strain and the deletion strains of Set2, Alp13 and the double knock-out of Hrp1 and Hrp3. Each experiment was performed twice in biological replicates All experiments were performed twice in biological replicates, except for the expression array of set2M-NM-^T. The replicates of hrp3M-NM-^T and hrp1M-NM-^Thrp3M-NM-^T were performed with a slightly different array design All experiments were performed twice in biological replicates. The replicates of hrp3M-NM-^T and hrp1M-NM-^Thrp3M-NM-^T were performed with a slightly different array design MNase treated sample were comparatively hybridized with genomic DNA of corresponding strain, WT, hrp1d hrp3d, pob3d in biological duplicates, set2d, alp13d, mit1d, hrp1d, hrp3d analysis was performed once

Dataset Information

CENP-B cooperates with Set1 in bidirectional transcriptional silencing and genome organization of retrotransposons

Publications

CENP-B cooperates with Set1 in bidirectional transcriptional silencing and genome organization of retrotransposons.

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