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. 2009 Apr 28;106(17):6956-61.
doi: 10.1073/pnas.0806302106. Epub 2009 Apr 13.

Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Karen Zhou  1 , Wei Hung William Kuo, Jeffrey Fillingham, Jack F Greenblatt
Affiliations

Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Karen Zhou et al. Proc Natl Acad Sci U S A. .

Abstract

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1-Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Yeast Spt5 is a substrate of the BUR kinase. (A) Sequence of the Spt5 C-terminal region. The region contains 15 repeats (underlined) of a hexapeptide whose consensus sequence is S[T/A]WGG[Q/A]. (B) BUR kinase is needed for phosphorylation of Spt5 in vivo. Spt5-TAP was immunoprecipitated from extracts of the indicated strains with IgG Sepharose and the precipitates were Western-blotted with the indicated antibodies. (C) BUR kinase can directly phosphorylate the Spt5 CTR in vitro. (D) Bur2-TAP, Ctk1-TAP, and Kin28-TAP were purified from TAP-tagged yeast strains and used, as indicated, to phosphorylate chicken core histones and GST-Spt5 CTR fusion protein in vitro.
Fig. 2.
Fig. 2.
The Spt5 CTR is important for growth and promotes transcription elongation. (A) A diploid strain was transformed with a 3xHA-His3MX6 cassette replacing either the entire C-terminal region (repeats 1–15) of Spt5 or only part of the CTR (repeats 10–15). After sporulation, tetrads were dissected and individual spores were tested for growth. Left shows 2 representative tetrads from diploids in which all 15 repeats were removed. Right shows 2 representative tetrads from diploids in which 6 of 15 repeats were removed. The arrows point to the 2 spores (meiotic products) of each tetrad that segregate with the CTR deletion. (B) Deletion of the Spt5 CTR causes sensitivity to elongation inhibitors. Ten-fold serial dilutions of SPT5 and spt5ctrΔ(–15) strains were tested for growth on plates in the presence of mycophenolic acid (MPA) or 6-AU. (C) Representative chromatin immunoprecipitation (ChIP) showing that elongation by RNAPII is less efficient when the Spt5 CTR is deleted. Chromatin was prepared from SPT5 and spt5 CTRΔ(–15) strains grown in the presence or absence of 6-AU, as indicated. Chromatin was precipitated with 8WG16 monoclonal antibody that recognizes RNAPII and analyzed using primers specific for the PMA1 locus. A diagram of the PMA1 locus is shown to indicate the location of the ChIP primer pairs (from ref. 56). The asterisks indicate a nontranscribed sequence near the telomere of chromosome 5 that is coamplified in each PCR as a control for nonspecific precipitation. IN, input DNA; IP, precipitated DNA. (D) Quantification of ChIP data like that shown in Fig. 2C, with error bars indicating standard deviations. Relative enrichment was calculated as [(Experimental band IP)/(Control band IP)]/[(Input Experimental band)/(Input Control band)]. Promoter, cds1,2,3, and 3′UTR correspond to primer pairs 1, 2, 3, 4, and 6, respectively.
Fig. 3.
Fig. 3.
Cotranscriptional histone modification depends on the Spt5 CTR. All strains contained FLAG-tagged histone H2B and were derived from strain YZS246 (34). Whole-cell extracts were Western-blotted with either M2 anti-FLAG antibody (top gel) or the indicated antibodies.
Fig. 4.
Fig. 4.
The Spt5 C-terminal domain is required for optimal recruitment of the PAF complex. (A) Recruitment of the PAF complex to PMA1is reduced in the spt5 CTRΔ(–15) mutant. ChIP was performed with anti-TAP antibody on the indicated strains containing TAP tags on the Rtf1 subunit of the PAF complex. A diagram of the PMA1 locus is shown to indicate location of the ChIP primer pairs (from ref. 56). The asterisks indicate a nontranscribed sequence near the telomere of chromosome 5 that is coamplified in each PCR as a control for nonspecific precipitation. IN, input DNA; IP, precipitated DNA. (B) Quantification of ChIP data like that shown in Fig. 2B, with error bars indicating standard deviations. Promoter, cds1,2,3,4, and 3′UTR correspond to primer pairs 1–6, respectively. (C) The Spt5 CTR is not required for the expression or stability of Rtf1. Yeast whole cell extracts from the indicated strains containing HA tags on Spt5 were Western blotted with anti-HA antibody, which also binds to the protein A moiety of the TAP tag on Rtf1 in these strains. (D) The Spt5 CTR is not required for recruitment of Spt5. ChIP was performed with an anti-HA antibody on chromatin prepared from yeast strains with SPT5-HA or spt5 CTRΔ(–15)-HA. Calculated enrichment scores are shown below each lane.
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