A unified view of the sequence and functional organization of the human RNA polymerase II promoter

Abstract

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5′ and 3′ ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

Nuclear export restricts Gdown1 to a mitotic function

“…Genomic coverage of pause and gene body regions for Parental and Gdown1 KO #3 PRO-Seq datasets were quantified using the bedtools coverage program and compared using the DESeq2 program. The tsrFinderM1 algorithm was applied to our HeLa NasCap data ( 43 ) with default parameters to discover TSRs and their respective MaxTSSs. The bedtools coverage program was used to calculate total fragments in 500 bp genomic intervals centered on MaxTSSs from our HeLa H3K4me1 and H3K4me3 datasets ( 40 ).…”

Quantitative analysis of transcription start site selection inSaccharomyces cerevisiaereveals control by DNA sequence, RNA Polymerase II activity, and NTP levels

“…First, in promoter scanning, the TSS and adjacent bases interact with Pol II active site, the 1 st NTP or each other to facilitate stable binding of 1 st NTP and potentially the 2 nd NTP to stimulate RNA synthesis. This would be in contrast to the concept of the initiator or downstream elements functioning as part of the TFIID or PIC binding site as has been proposed for higher eukaryotes (see (Luse, Parida, Spector, Nilson, & Price, 2020; Vo ngoc, Huang, Cassidy, Medrano, & Kadonaga, 2020) and references therein). As the universal initiating element, Y-1R+1 has been established to facilitate stable binding of the NTPs by RNA polymerases via base stacking between R-1 from template DNA and the 1 st purine NTP (Basu et al, 2014;Gleghorn et al, 2011).…”

Ssl2/TFIIH function in transcription start site scanning by RNA polymerase II in Saccharomyces cerevisiae

“…Therefore, whether or not scanning is also a mechanism in higher eukaryotic promoters, or minimally for a subset of eukaryotic promoters or within a specified window, is still an unanswered question as there have been no formal tests of this mechanism. It has been suggested, however, that each individual TSS is recognized as an individual promoter due to sequence signatures apparent in comparison of thousands of TSSs in humans ( Luse et al, 2020 ). Very recent results of cryo-EM studies on human PICs, especially in structures visualizing TFIID, indicate that promoter classes may assemble PIC components in distinct fashion within a single organism ( Chen et al, 2021 ), yet these assembly pathways similarly position an upstream TSS proximal to the Pol II active site, consistent with proposals that human promoters could contain information for assembling PICs individually ( Luse et al, 2020 ).…”

“…It has been suggested, however, that each individual TSS is recognized as an individual promoter due to sequence signatures apparent in comparison of thousands of TSSs in humans ( Luse et al, 2020 ). Very recent results of cryo-EM studies on human PICs, especially in structures visualizing TFIID, indicate that promoter classes may assemble PIC components in distinct fashion within a single organism ( Chen et al, 2021 ), yet these assembly pathways similarly position an upstream TSS proximal to the Pol II active site, consistent with proposals that human promoters could contain information for assembling PICs individually ( Luse et al, 2020 ). Recent results suggest that there may be plasticity in TSS selection from individual PICs upon mutation of Inr sites in mouse, potentially supporting a type of scanning in mammals ( Chou et al, 2021 ).…”