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Abstract
Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD ‘code’ for co-transcriptional histone modifications.
Highlights
The RNA polymerase II (RNAPII) elongation complex coordinates transcription with key co-transcriptional events such as mRNA processing and histone post-translational modification
Rpb1 and Spt5 have distinctive C-terminal domains (CTDs) comprising multiple repeats of a short motif; their CTDs interact with multiple regulators and are critical for assembly of a functional elongation complex [1,2]
Multiple components of the RNAPII elongation complex can bind to both CTD domains, and genetic analysis indicates that the two CTDs have overlapping functions in vivo [1,2,5,8,9,10]
Summary
The RNA polymerase II (RNAPII) elongation complex coordinates transcription with key co-transcriptional events such as mRNA processing and histone post-translational modification. The Rpb and Spt CTDs direct co-transcriptional histone modifications, which are present in a stereotyped pattern within the coding regions of transcribed genes. The PAF complex, a multi-functional elongation factor that promotes H2Bub, H3K4me and H3K36me, interacts with diphosphorylated Rpb S2-P/S5-P peptides as well as with Spt T1-P. These interactions involve the PAF subunits Ctr and Cdc73 [26,27]. H3K36me depends on Cdk in the nematode Caenorhabditis elegans as well [43] While these studies have highlighted the importance of some combination of Rpb S5-P, Rpb S2-P and Spt T1-P for co-transcriptional histone modifications, they do not address the contributions of individual sites and how the sites functionally interact. Our findings provide novel insights into the mechanisms linking the histone and CTD modification programs
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