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

ABSTRACT 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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Available from: Michael Christopher Keogh, Sep 28, 2015
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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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    Cell Reports 02/2014; 6(5). DOI:10.1016/j.celrep.2014.01.029 · 8.36 Impact Factor
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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.
    Nucleic Acids Research 01/2014; 42(7). DOI:10.1093/nar/gkt1398 · 9.11 Impact Factor
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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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