Project description:Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although viewed as an elongation factor, spt6 mutations allow transcription from within coding regions, suggesting that Spt6 also controls initiation. To comprehensively characterize the requirement for Spt6 in transcription, we have used four approaches: TSS-seq and TFIIB ChIP-nexus to assay transcription initiation, NET-seq to assay elongating RNAPII, and MNase-seq to assay nucleosome occupancy and positioning. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters, and find some features conserved with genic promoters and other features that are distinct. Finally, we show that Spt6 regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome and reveal the magnitude of the potential for expressing alternative genetic information via intragenic promoters.
Project description:Nucleosome positioning is both active and passive and regulates access to the genome for replication, transcription and repair. Here we report that Mit1, a subunit of the fission yeast SHREC complex similar to Mi-2/NuRD, regulates transcription at regions of heterochromatin by positioning nucleosomes to preclude access to RNA Polymerase II. Purified Mit1 is a nucleosome remodeling factor capable of mobilizing histone octamers on short DNA fragments and requires ATP hydrolysis and chromatin tethering domains to remodel nucleosomes and silence transcription. We propose that SHREC is recruited to heterochromatin to mobilize nucleosomes onto unfavorable positions to prevent spurious transcription within heterochromatin. M-bM-^@M-^C RNA samples were prepared from biological duplicates of WT and mit1M-bM-^HM-^F::NatMX6 fission yeast to compare transcript levels using the Affymetrix GeneChip S.pombe Tiling 1.0FR microarray.
Project description:Nucleosome positioning is both active and passive and regulates access to the genome for replication, transcription and repair. Here we report that Mit1, a subunit of the fission yeast SHREC complex similar to Mi-2/NuRD, regulates transcription at regions of heterochromatin by positioning nucleosomes to preclude access to RNA Polymerase II. Purified Mit1 is a nucleosome remodeling factor capable of mobilizing histone octamers on short DNA fragments and requires ATP hydrolysis and chromatin tethering domains to remodel nucleosomes and silence transcription. We propose that SHREC is recruited to heterochromatin to mobilize nucleosomes onto unfavorable positions to prevent spurious transcription within heterochromatin.
Project description:The positioning of the nucleosome by ATP-dependent nucleosome remodelers provides the fundamental chromatin environment for the regulation of diverse cellular processes acting on the underlying DNA. Here, we report that the fission yeast CHD remodeler, Hrp3, is a global regulator that drives higher-order chromatin structure and genomic stability. The loss of Hrp3 resulted in the production of antisense transcripts and perturbation of the nucleosome in an ATPase mutant of Hrp3 was also associated with destabilization of the DNA-histone interaction. Furthermore, the effect of Hrp3 in the pericentric region was found to be accomplished via a physical interaction with Swi6, and appeared to cooperate with other heterochromatin factors for gene silencing. Taken together, our data indicate that a well-positioned nucleosome is important for the spatial-temporal control of transcription-associated processes, and show that this is accomplished by the chromatin remodeler, Hrp3, in fission yeast. Two-condition experiment, Mutant vs Wild-type (control)
Project description:CK2 is an essential protein kinase implicated in various cellular processes. In this study, we address a potential role of this kinase in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells leads to replication-independent increase of histone H3K56 acetylation and global activation of H3 turnover in coding regions. This suggests a positive role of CK2 in maintenance/recycling of the histone H3/H4 tetramers during transcription. Interestingly, strand-specific RNA-seq analyses show that CK2 inhibits global cryptic promoters driving both sense and antisense transcription. This further indicates a role of CK2 in the modulation of chromatin during transcription. Next, we showed that CK2 interacts with the major histone chaperone Spt6, and phosphorylates it in vivo and in vitro. CK2 phosphorylation of Spt6 is required for its cellular levels, for the suppression of histone H3 turnover and for the inhibition of spurious transcription. Finally, we show that CK2 and Spt6 phosphorylation sites are important to various transcriptional responses suggesting that cryptic intragenic and antisense transcript production may have an impact on cell adaptation to environmental cues. Altogether, our data indicate that CK2 mediated phosphorylation of Spt6 regulates chromatin dynamics associated with transcription, and prevents aberrant transcription.
Project description:CK2 is an essential protein kinase implicated in various cellular processes. In this study, we address a potential role of this kinase in chromatin modulations associated with transcription. We found that CK2 depletion from yeast cells leads to replication-independent increase of histone H3K56 acetylation and global activation of H3 turnover in coding regions. This suggests a positive role of CK2 in maintenance/recycling of the histone H3/H4 tetramers during transcription. Interestingly, strand-specific RNA-seq analyses show that CK2 inhibits global cryptic promoters driving both sense and antisense transcription. This further indicates a role of CK2 in the modulation of chromatin during transcription. Next, we showed that CK2 interacts with the major histone chaperone Spt6, and phosphorylates it in vivo and in vitro. CK2 phosphorylation of Spt6 is required for its cellular levels, for the suppression of histone H3 turnover and for the inhibition of spurious transcription. Finally, we show that CK2 and Spt6 phosphorylation sites are important to various transcriptional responses suggesting that cryptic intragenic and antisense transcript production may have an impact on cell adaptation to environmental cues. Altogether, our data indicate that CK2 mediated phosphorylation of Spt6 regulates chromatin dynamics associated with transcription, and prevents aberrant transcription.