Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types

Background: For many genes, RNA polymerase II stably pauses before transitioning to productive elongation. Although polymerase II pausing has been shown to be a mechanism for regulating transcriptional activation, the extent to which it is involved in control of mammalian gene expression and its relationship to chromatin structure remain poorly understood. Results: Here, we analyze 85 RNA polymerase II chromatin immunoprecipitation (ChIP)-sequencing experiments from 35 different murine and human samples, as well as related genome-wide datasets, to gain new insights into the relationship between polymerase II pausing and gene regulation. Across cell and tissue types, paused genes (pausing index > 2) comprise approximately 60 % of expressed genes and are repeatedly associated with specific biological functions. Paused genes also have lower cell-to-cell expression variability. Increased pausing has a non-linear effect on gene expression levels, with moderately paused genes being expressed more highly than other paused genes. The highest gene expression levels are often achieved through a novel pause-release mechanism driven by high polymerase II initiation. In three datasets examining the impact of extracellular signals, genes responsive to stimulus have slightly lower pausing index on average than non-responsive genes, and rapid gene activation is linked to conditional pause-release. Both chromatin structure and local sequence composition near the transcription start site influence pausing, with divergent features between mammals and Drosophila. Most notably, in mammals pausing is positively correlated with histone H2A.Z occupancy at promoters. Conclusions: Our results provide new insights into the contribution of RNA polymerase II pausing in mammalian gene regulation and chromatin structure. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0984-2) contains supplementary material, which is available to authorized users

Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP

The conserved histone locus body (HLB) assembles prior to zygotic gene activation early during development and concentrates factors into a nuclear domain of coordinated histone gene regulation. Although HLBs form specifically at replication-dependent histone loci, the cis and trans factors that target HLB components to histone genes remained unknown. Here we report that conserved GA repeat cis elements within the bidirectional histone3–histone4 promoter direct HLB formation in Drosophila. In addition, the CLAMP (chromatin-linked adaptor for male-specific lethal [MSL] proteins) zinc finger protein binds these GA repeat motifs, increases chromatin accessibility, enhances histone gene transcription, and promotes HLB formation. We demonstrated previously that CLAMP also promotes the formation of another domain of coordinated gene regulation: the dosage-compensated male X chromosome. Therefore, CLAMP binding to GA repeat motifs promotes the formation of two distinct domains of coordinated gene activation located at different places in the genome

List of proteins identified in all three mass spectrometry approaches.

<p>List of proteins identified in all three mass spectrometry approaches.</p

The <i>Drosophila</i> CLAMP protein associates with diverse proteins on chromatin

<div><p>Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. We previously identified a zinc finger protein in <i>Drosophila melanogaster</i> that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). More recently, we established that CLAMP has non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, we used two complementary mass spectrometry approaches. We demonstrate that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, we have identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.</p></div

CLAMP inhibits NELF recruitment to highly paused genes.

<p>Chromatin immunoprecipitation of NELF-B was performed from S2 cells treated with either <i>gfp</i> control (blue) or <i>clamp</i> (green) RNAi. The values for log₂-fold enrichment over Input are shown after normalizing internally to a control locus (<i>cg15570</i>) that is unbound for CLAMP or NELF-B. These values were then normalized to Input to generate the log₂-fold enrichment value. Three separate biological replicates were averaged and the standard error of the mean was calculated (error bars are +/- 1 S.E.M.). Significance was determined using Kruskal-Wallis test by ranks, where the asterisk indicates a p-value <0.05. The y-axis on the left shows enrichment scores for the low paused genes, while the y-axis on the right indicates the values for the high paused genes. The highly paused genes have greater enrichment of NELF-B than the lowly paused genes, as expected.</p

Overlap and distribution relative to genes of CLAMP, GAF, and NELF ChIP-seq occupancy.

<p>Comparison of CLAMP and GAF ChIP-seq peaks with NELF-B ChIP-chip peaks was performed to determine the overlap between factor occupancy. The numbers in dark green represent the percentage of CLAMP peaks, dark red are the percentage of GAF peaks, and dark blue are the percentage of NELF peaks. (A) Only 43% of CLAMP peaks overlap with GAF, whereas the majority (82%) of GAF peaks overlap with CLAMP. (B) A small fraction (11%) of CLAMP peaks overlap with NELF, while most NELF peaks also contain CLAMP (72%). (C) Fewer NELF peaks (67%) associate with GAF than with CLAMP (72%). (D) Venn diagram describes the percentage of CLAMP, GAF, and NELF peaks that overlap with each of the other factors. (E) CLAMP, GAF, and NELF peaks were categorized as either within +/- 250bp centered on the transcription start site (TSS), between +250bp and the end of the gene (gene body), or intergenic (all other peaks). The percentages of total CLAMP, GAF, or NELF peaks that fall within each of these regions are in the first three rows. Next, the distribution of CLAMP peaks that are also occupied by NELF, GAF, or both is shown in the last three rows.</p

CLAMP does not regulate NELF-B protein abundance.

<p>(A) Transcript abundance for <i>clamp</i> and <i>nelf-b</i> was measured to determine the difference in transcript abundance between control (<i>gfp</i>, blue) and <i>clamp</i> (green) RNAi treatment by qPCR. The average fold change (ΔCt compared to <i>gapdh</i>) from four biological replicates is shown for both <i>clamp</i> and <i>nelf-b</i> transcripts. As expected, abundance of <i>clamp</i> is reduced after <i>clamp</i> RNAi, while <i>nelf-b</i> transcript abundance is not affected compared to the control RNAi. The error bars represent +/- 1 S.D., with p-values indicated. (B) Protein accumulation was measured by western blot for both CLAMP and NELF-B after of <i>clamp</i> and <i>gfp</i> control RNAi. RNAi targeting <i>clamp</i> reduces the amount of CLAMP protein but has no effect on NELF-B protein levels. Actin is used as a loading control.</p