Abstract
The RNA polymerase II (RNAPII) associated factor 1 complex (Paf1C) plays critical roles in modulating the release of paused RNAPII into productive elongation. However, regulation of Paf1C-mediated promoter-proximal pausing is complex and context dependent. In fact, in cancer cell lines, opposing models of Paf1Cs' role in RNAPII pause-release control have been proposed. Here, we show that the Paf1C positively regulates enhancer activity in mouse embryonic stem cells. In particular, our analyses reveal extensive Paf1C occupancy and function at super enhancers. Importantly, Paf1C occupancy correlates with the strength of enhancer activity, improving the predictive power to classify enhancers in genomic sequences. Depletion of Paf1C attenuates the expression of genes regulated by targeted enhancers and affects RNAPII Ser2 phosphorylation at the binding sites, suggesting that Paf1C-mediated positive regulation of pluripotency enhancers is crucial to maintain mouse embryonic stem cell self-renewal.
Highlights
Transcription of many eukaryotic protein-coding genes is regulated in large part by enhancers, DNA sequences that increase the likelihood that transcription of a particular gene will occur under favorable conditions [1]
To elucidate molecular functions of Paf1C, we generated a GFPtagged knock-in mouse embryonic stem cells (mESCs) cell line, where GFP is fused to the 39 terminus of Ctr9, a core component of the Paf1C
Analyses of the ChIP-seq experiments identified 33,412 and 13,471 cell type–specific Ctr9 binding sites in mESCs and NIH3T3 cells, respectively, whereas 5,673 common binding sites were identified in both cell types (Table S1). 76% of the Ctr9 binding sites were found at protein-coding genes, whereas 24% of the binding sites mapped to intergenic region in mESCs (Fig S2A)
Summary
Transcription of many eukaryotic protein-coding genes is regulated in large part by enhancers, DNA sequences that increase the likelihood that transcription of a particular gene will occur under favorable conditions [1]. Enhancers are found in intergenic regions, introns and exons, and can activate transcription independently of their location, distance, or orientation with respect to promoters [2]. A range of methods have been developed to predict enhancers, based on their characteristics including transcription factor binding, chromatin accessibility, histone modifications, or promoter–enhancer interactions. None of these methods correlate perfectly with enhancer activity because most active enhancers carry only partial characteristic marks (summarized in reference 3). In contrast to the indirect prediction methods, a recently developed technique named self-transcribing active regulatory region sequencing (STARR-seq) allows direct survey of active enhancers by coupling enhancer activity to its sequence in cis. STARR-seq has been applied genome-wide in flies and in some mammalian cells and has greatly advanced our understanding of how enhancer activities are encoded in the genome [4, 5]
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