The Replication-independent Histone H3-H4 Chaperones HIR, ASF1, and RTT106 Co-operate to Maintain Promoter Fidelity

Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2011; 287(3):1709-18. DOI: 10.1074/jbc.M111.316489
Source: PubMed


RNA polymerase II initiates from low complexity sequences so cells must reliably distinguish "real" from "cryptic" promoters and maintain fidelity to the former. Further, this must be performed under a range of conditions, including those found within inactive and highly transcribed regions. Here, we used genome-scale screening to identify those factors that regulate the use of a specific cryptic promoter and how this is influenced by the degree of transcription over the element. We show that promoter fidelity is most reliant on histone gene transactivators (Spt10, Spt21) and H3-H4 chaperones (Asf1, HIR complex) from the replication-independent deposition pathway. Mutations of Rtt106 that abrogate its interactions with H3-H4 or dsDNA permit extensive cryptic transcription comparable with replication-independent deposition factor deletions. We propose that nucleosome shielding is the primary means to maintain promoter fidelity, and histone replacement is most efficiently mediated in yeast cells by a HIR/Asf1/H3-H4/Rtt106 pathway.

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    • "ChIP samples were prepared as above and qPCR data obtained with an iCycler (Bio-Rad), SYBR green (Molecular Probes), Platinum Taq (Invitrogen), and the primer sets in Table S3. Quantitations were as previously described (Silva et al., 2012), with all enrichments expressed as a percentage of input or relative to a gene-free region (GFR: Chromosome II 1,149,380–1,149,504) that shows no specific enrichment of TBP, Nrc1, Cka1, Taf7, or Cut3 in ChIP-seq (Figure S3). "
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    ABSTRACT: Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.
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    • "The integrity of the repressive chromatin is also maintained by the histone H3K36 methyltransferase Set2p, which recruits the Rpd3S histone deacetylase to remove transcription elongation-associated acetylation (7,9). Likewise, alterations in transcription-dependent H3–H4 deposition by mutating factors in the HIR/Asf1p/Rtt106p pathway (5,8,12) also result in spurious intragenic transcripts. "
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    ABSTRACT: Chromatin structure in transcribed regions poses a barrier for intragenic transcription. In a comprehensive study of the yeast chromatin remodelers and the Mot1p-NC2 regulators of TATA-binding protein (TBP), we detected synthetic genetic interactions indicative of suppression of intragenic transcription. Conditional depletion of Mot1p or NC2 in absence of the ISW1 remodeler, but not in the absence of other chromatin remodelers, activated the cryptic FLO8 promoter. Likewise, conditional depletion of Mot1p or NC2 in deletion backgrounds of the H3K36 methyltransferase Set2p or the Asf1p-Rtt106p histone H3-H4 chaperones, important factors involved in maintaining a repressive chromatin environment, resulted in increased intragenic FLO8 transcripts. Activity of the cryptic FLO8 promoter is associated with reduced H3 levels, increased TBP binding and tri-methylation of H3K4 and is independent of Spt-Ada-Gcn5-acetyltransferase function. These data reveal cooperation of negative regulation of TBP with specific chromatin regulators to inhibit intragenic transcription.
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    • "Collectively, these data show that the PAF complex interacts with, and may regulate the function of Spt6. Consistent with regulation of Spt6 and Ser2 CTD phosphorylation by PAF, and in agreement with recent reports (54,61), we found that the deletions of PAF1 and CTR9 display a cryptic transcription phenotype (Figure 4E). We also showed that PAF member deletions are sensitive to 6-AU, thus confirming the role of this complex in promoting transcription elongation in addition to maintaining chromatin integrity (Figure 4F). "
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    ABSTRACT: The C-terminal domain (CTD) of RNA polymerase II is sequentially modified for recruitment of numerous accessory factors during transcription. One such factor is Spt6, which couples transcription elongation with histone chaperone activity and the regulation of H3 lysine 36 methylation. Here, we show that CTD association of Spt6 is required for Ser2 CTD phosphorylation and for the protein stability of Ctk1 (the major Ser2 CTD kinase). We also find that Spt6 associates with Ctk1, and, unexpectedly, Ctk1 and Ser2 CTD phosphorylation are required for the stability of Spt6—thus revealing a Spt6–Ctk1 feed-forward loop that robustly maintains Ser2 phosphorylation during transcription. In addition, we find that the BUR kinase and the polymerase associated factor transcription complex function upstream of the Spt6–Ctk1 loop, most likely by recruiting Spt6 to the CTD at the onset of transcription. Consistent with requirement of Spt6 in histone gene expression and nucleosome deposition, mutation or deletion of members of the Spt6–Ctk1 loop leads to global loss of histone H3 and sensitivity to hydroxyurea. In sum, these results elucidate a new control mechanism for the regulation of RNAPII CTD phosphorylation during transcription elongation that is likely to be highly conserved.
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