The Importance of Controlling Transcription Elongation at Coding and Noncoding RNA Loci

Abstract

Here we discuss current paradigms for how transcription initiation and elongation control are achieved in mammalian cells, and how they differ at protein-coding mRNA genes versus noncoding RNA (ncRNA) loci. We present a model for the function of ncRNAs wherein the act of transcription is regulatory, rather than the ncRNA products themselves. We further describe how the establishment of transcriptionally engaged, but paused, RNA polymerase II impacts chromatin structure around divergent transcription start sites, and how this can influence transcription factor binding and mRNA gene activity in the region.

Figure 1.. Overview of transcription cycle.

A. Pioneer factors (blue, PF) bind their DNA motif, even in the presence of a nucleosome. B. In cooperation with other transcription factors (yellow, TF), pioneer factors recruit chromatin remodelers to expose core promoter elements (brown) near TSSs. C. General transcription factors (brown) and Mediator associate with the core promoter and TFs, enabling recruitment of Pol II and formation of a pre-initiation complex. D. Pause-inducing factors NELF and DSIF bind the early elongation complex to stably pause RNA synthesis. E. P-TEFb recruitment, often within the Super Elongation Complex (SEC) leads to phosphorylation of DSIF causing dissociation of NELF and pause release. F. Productive elongation of Pol II into the gene body is associated with loading of chromatin modifying factors and RNA processing factors like the U1 snRNP.

Figure 2.. Core promoter elements help recruit and position Pol II

A. A number of core promoter motifs have been defined, including upstream and downstream TFIIB recognition elements (BRE^u and BRE^d, respectively; dark red), TATA box (yellow), Initiator element (Inr, green), downstream core elements (DCE1–3; blue), motif ten element (MTE, purple), and downstream promoter element (DPE, red). Subunits of general transcription factors TFIIB and TFIID bind the core elements. Positions are shown relative to the TSS. B. Typically, only a subset of core promoter elements are found at TSSs used in vivo, with no single promoter element being required for transcription initiation.

Figure 3.. Transcription initiation is often highly focused.

Examples of promoters defined as (A) focused, (B) intermediate, and (C) dispersed (definitions are based on Rach et al. 2011). Mouse promoters were classified using Start-seq reads from macrophages, in a window ±50 nt from the peak read location, which we call the ‘observed TSS’. D. Similarly focused transcription initiation is observed across gene classes.

Figure 4.. Upstream anti-sense transcripts generally terminate close to the TSS.

A. Divergent promoters consist of two distinct TSSs oriented in opposing directions. Pol II pausing occurs downstream of both the mRNA and uaRNA TSSs, allowing for detection of nascent Start-RNAs. B. RNA synthesis extends beyond 200 nt predominantly in the sense direction, resulting in the transcription of a protein-coding mRNA. Elongation in the opposite direction is typically non-processive, generating a short ncRNA that is subject to degradation. C. Similar increases in early elongation complexes (measured by Start-RNA levels at the TSS ± 100 bp) were observed for the mRNA and uaRNAs of early response genes following 30 minute activation with bacterial LPS (left). By comparison, evaluation of longer nascent RNAs (Bhatt et al. 2012; window from TSS to +250 bp) reveals that productive elongation is biased heavily towards the sense direction (right).

Figure 5.. U1 and PAS motifs influence elongation properties

A. The cumulative percentage of genes containing motifs recognized by the U1 snRNP are plotted relative to anti-sense (left) or sense (right) TSSs. Distance to the closest exon-intron junction (5’ splice sites; orange) are also shown for sense TSSs. The dashed red line indicates 200nt downstream from each TSS, to indicate where a decision concerning processivity is likely to occur. B. The cumulative percentage of genes containing poly-A sequences (PAS) are plotted relative to the anti-sense (left) or sense (right) TSS as in (A).

Figure 6.. Variable promoter chromatin architectures reflect variable distances between divergent TSSs. Modified from Scruggs et al. 2015.

A. Start-RNA reads from mouse macrophages are shown in the anti-sense (purple) and sense (green) direction, both aligned with respect to sense TSSs. Genes are rank ordered by the distance between sense and anti-sense TSSs. B. Pol II and TBP ChIP-seq, MNase-seq, and FAIRE-seq are shown for TSSs organized as in (A).

Figure 7.. Coordination between core promoter elements and TF motifs

A. Heat maps depict Start-RNAs around macrophage anti-sense (purple) and sense (green) TSSs, as in Figure 6. Shown are a panel of vertebrate TF motifs (red), and Inr motifs on the anti-sense (purple) or sense (green) strand. Combined vertebrate TF and Inr motifs are shown at right. Lower heat maps show genes lacking defined anti-sense TSS. We note that these genes are not devoid of anti-sense Start-RNA reads. B. Average distribution of vertebrate TF motifs relative to anti-sense TSSs (left) and sense TSSs (right). TF motif occurrence peaks next to each TSS. C. Distribution of Inr motifs (black) and observed uaRNA TSS positions (dashed purple) upstream of the mRNA TSS, plotted as a cumulative total. Genes are those with divergent TSSs (left) or without clearly defined uaRNA TSSs (right).

Figure 8.. Establishment of divergent promoters is a multistep process involving coordination between transcription factors and Pol II.

A. Pioneer factors (PF) recognize their DNA binding motifs near nucleosome-occluded TSSs. B. Cooperation between TFs opens chromatin and recruits remodelers, exposing core promoter elements. C. Initiation and pausing of Pol II helps maintain open chromatin, establishing a nucleosome-depleted region between divergent TSSs. D. Chromatin modifying factors associated with elongating Pol II establish distinct epigenetic landscapes downstream of divergent TSSs. E. Maintenance of open chromatin renders motifs for binding of signal-dependent transcription factors (SDF, orange) accessible for immediate binding during gene activation.