PAF1, a Molecular Regulator of Promoter-Proximal Pausing by RNA Polymerase II

The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II's C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II

Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

<div><p>The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.</p></div

TBPH and Lark depletion alter Nipped-B and cohesin occupancy at gene regulatory sequences in BG3 cells.

<p>The browser tracks show the log2 enrichment for TBPH, Lark, Nipped-B, SA, and Nipped-B and SA after RNAi depletion of TBPH (iTBPH) and Lark (iLark) for the <i>string</i> gene and its upstream enhancers. The <i>string</i> enhancers that are most resistant to decreases in Nipped-B and SA occupancy upon TBPH depletion are marked with asterisks. The tracks shown are the average of two to five independent biological replicates. The boxplots show the distributions of the average log2 enrichment for all active promoters (gray), extragenic enhancers (yellow) and PREs (orange) in mock-treated (Mock) cells, and cells in which TBPH (iTBPH) or Lark (iLark) have been depleted by RNAi for four to five days. Gene regulatory elements are defined as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.g002" target="_blank">Fig 2</a>. Asterisks indicate distributions that differ significantly from the mock control using a paired t test. The p values are given in the main text. A paired t test was used because each regulatory sequence is matched with itself in the control and depleted cells and the distributions are close to normal. All comparisons indicated as significant are also significant using the non-parametric Wilcoxon signed rank test. Plots of the values for all individual regulatory sequences in mock control versus the depleted cells are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s006" target="_blank">S6 Fig</a>.</p

TBPH and Lark preferentially bind RNAs from cohesin-binding genes in vitro using known RNA-binding domains.

<p>Diagrams of the TBPH and Lark protein structures with known sequence domains shown at the top: RRM, RNA Recognition Motif; ZnF, Zinc Finger. The smallest fragments tested that bind RNA in vitro are underlined in red. The bar graphs below the protein diagrams show examples of RNA-binding competition experiments with TBPH (left) and Lark (right). Whether or not the RNA is from a cohesin-binding gene or contains UG repeats is indicated (Y = yes, N = no). The sequences of all short RNAs tested from cohesin-binding and non-binding genes are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s013" target="_blank">S1 Table</a>, along with a summary of their abilities to bind TBPH and Lark derived from multiple experiments. As detailed in the text, soluble His₆-SUMO fusion proteins were immobilized on NTA-Zn²⁺ agarose beads and incubated with equimolar mixtures of the indicated short RNAs. RNAs that were retained after washing were quantified by real-time PCR and binding was defined by enrichment relative to the amount of the CG6310-3 control RNA, which does not bind to either protein. All RNA fragments were tested in two independent binding experiments with freshly made fusion proteins, and each RNA was measured twice in each experiment. Error bars are the standard deviations of all measurements in all experiments. Enrichment of 10-fold or greater is defined as specific binding when recorded in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s013" target="_blank">S1 Table</a>. The bottom bar graphs show example RNA-binding experiments with the indicated fragments of TBPH and Lark to map the domains that bind RNA. SDS-PAGE characterization of the immobilized protein fragments and the residues contained within each fragment are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s010" target="_blank">S10 Fig</a>. For truncated proteins, RNA enrichment greater than or equal to half the enrichment obtained with the full-length protein in the same experiment was defined as binding. With TBPH, the RRM1-containing region is necessary and sufficient for RNA-binding. For Lark, the zinc finger (ZnF) is required in addition to the RRM-containing domain. It is unknown if one or both of the Lark RRM domains is required to bind RNA.</p

Nipped-B facilitates binding of TBPH and Lark to gene regulatory sequences in BG3 cells.

<p>The browser view at the upper left shows log2 ChIP-seq enrichment tracks for Nipped-B in control cells, and for TBPH and Lark in control cells and cells depleted for Nipped-B (iNipped-B) at the <i>string</i> gene and its enhancers. The boxplots at the upper right show the distributions of ChIP-seq enrichment for TBPH and Lark at active promoters (PRO) extragenic enhancers (ENH) and PREs (PRE) in control (Mock) cells and cells depleted for Nipped-B (iNipped-B). Four biological replicates were averaged for the TBPH ChIP-seq and two for Lark ChIP-seq in Nipped-B depleted cells. Distributions that differ significantly from the Mock control by the paired t test are marked with asterisks, and the p values are given in the main text. All marked comparisons are also significant by the Wilcoxon signed rank test. For each regulatory sequence, the log2 enrichment values in Mock control cells (Mock log2 enrichment) are plotted against the enrichment values in cells depleted for Nipped-B (iNipped-B log2 enrichment) in the dot plots.</p

Hypothetical models for roles of TBPH and Lark in Nipped-B and cohesin binding to genes and enhancers, enhancer-promoter interactions, and processing of nascent RNA transcripts.

<p>At promoters (top row) we posit that TBPH binds to UG repeats in the first nascent transcripts produced by elongating Pol II (Ser2P / Ser5P) when a gene is initially activated. TBPH then recruits Nipped-B, which interacts with DNA, loads cohesin and recruits the Lark RNA-binding protein. At enhancers (middle row) activator proteins (purple oval) recruit Mediator (large tan circle) and Nipped-B. Nipped-B then loads cohesin and recruits TBPH and Lark, and TBPH stabilizes binding of the complex. The protein complexes at the enhancers and promoters form enhancer-promoter complexes (bottom row) that are stable for hours even in the absence of new transcription initiation. TBPH contributes to their stability, while Lark destabilizes cohesin and Nipped-B binding, particularly to the promoter. P-TEFb in the enhancer-promoter complex phosphorylates paused Pol II and the pausing factors (not depicted), leading to transcriptional elongation (lower right). Some TBPH and Lark present in the enhancer-promoter complex binds to the nascent RNA produced by the elongating polymerase and can facilitate RNA processing. For example, they can regulate intron removal by splicing as depicted in the lower right diagram, in addition to other processes.</p