Abstract
The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs.
Highlights
Diverse roles for Paf1CPaf1C has been intensively explored in yeast, flies, and mammalian cells, which has led to diverse, and sometimes differing, conclusions
Gene transcription can be regulated through multiple mechanisms, such as histone modifications that create structural changes of the chromatin leading to gene activation or suppression, or regulation of the 3’ cleavage site of the mRNA, known as alternative cleavage and polyadenylation (APA), resulting in the generation of transcript isoforms with various
By comparing depletions of two subunits, we show that the accumulation of RNA polymerase II (PolII) near the transcription start site (TSS) is associated with the activation of TSS-proximal pA sites observed in one depletion but not the other
Summary
Paf1C has been intensively explored in yeast, flies, and mammalian cells, which has led to diverse, and sometimes differing, conclusions. The complex was first characterized in yeast as a PolII-associated factor, and extensive use of mutants revealed that it plays a role in transcriptional elongation and chromatin modifications [1]. Mammalian Paf1C consists of six subunits (Paf, Cdc, Leo, Ctr, Rtf, and Ski8) [2, 3]. Very recent studies in flies and mammalian cells suggest that Paf could play a role in PolII pausing [6]. Human Paf, and to a lesser extent Leo, bind PolII, whereas Ski is more peripheral, and Rtf weakly associates with Paf1C [3, 7]. Promoter-bound trans-activators can recruit Paf1C [11]
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