Telmo Henriques

Stable Pausing by RNA Polymerase II Provides an Opportunity to Target and Integrate Regulatory Signals

Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.

Pausing of RNA polymerase II Regulates Mammalian Developmental Potential Through Control of Signaling Networks

The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

Widespread transcriptional pausing and elongation control at enhancers

Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.

The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning (‘intron definition’) or exon-spanning (‘exon definition’) pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60–70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.

Enhanced chromatin accessibility of the dosage compensated Drosophila male X-chromosome requires the CLAMP zinc finger protein

The essential process of dosage compensation is required to equalize gene expression of X-chromosome genes between males (XY) and females (XX). In Drosophila, the conserved Male-specific lethal (MSL) histone acetyltransferase complex mediates dosage compensation by increasing transcript levels from genes on the single male X-chromosome approximately two-fold. Consistent with its increased levels of transcription, the male X-chromosome has enhanced chromatin accessibility, distinguishing it from the autosomes. Here, we demonstrate that the non-sex-specific CLAMP (Chromatin-linked adaptor for MSL proteins) zinc finger protein that recognizes GA-rich sequences genome-wide promotes the specialized chromatin environment on the male X-chromosome and can act over long genomic distances (~14 kb). Although MSL complex is required for increasing transcript levels of X-linked genes, it is not required for enhancing global male X-chromosome chromatin accessibility, and instead works cooperatively with CLAMP to facilitate an accessible chromatin configuration at its sites of highest occupancy. Furthermore, CLAMP regulates chromatin structure at strong MSL complex binding sites through promoting recruitment of the Nucleosome Remodeling Factor (NURF) complex. In contrast to the X-chromosome, CLAMP regulates chromatin and gene expression on autosomes through a distinct mechanism that does not involve NURF recruitment. Overall, our results support a model where synergy between a non-sex-specific transcription factor (CLAMP) and a sex-specific cofactor (MSL) creates a specialized chromatin domain on the male X-chromosome.

Catching the waves: following the leading edge of elongating RNA polymerase II.

By precisely tracking the waves of elongating RNA polymerase II (Pol II) during gene activation, Danko et al. (2013), in this issue of Molecular Cell, discovered a surprising diversity of elongation rates among and along human genes.

NF-Y Controls Fidelity of Transcription Initiation at Gene Promoters Through Maintenance of the Nucleosome-Depleted Region

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription preinitiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results establish NF-Y as a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.

Transcriptional speed bumps revealed in high resolution

The enzyme RNA polymerase II, which transcribes DNA, pauses early in transcription and awaits signals to continue. High-resolution structures reveal how it is stopped and efficiently restarted.High-resolution structures of paused and active RNA polymerase II.

Intron Length and Recursive Sites are Major Determinants of Splicing Efficiency in Flies

The dynamics of gene expression may impact regulation, and RNA processing can be rate limiting. To assess rates of pre-mRNA splicing, we used a short, progressive metabolic labeling/RNA sequencing strategy to estimate the intron half-lives of ~30,000 fly introns, revealing strong correlations with several gene features. Splicing rates varied with intron length and were fastest for modal intron lengths of 60-70 nt. We also identified hundreds of novel recursively spliced segments, which were associated with much faster and also more accurate splicing of the long introns in which they occur. Surprisingly, the introns in a gene tend to have similar splicing half-lives and longer first introns are associated with faster splicing of subsequent introns. Our results indicate that genes have different intrinsic rates of splicing, and suggest that these rates are influenced by molecular events at gene 5’ ends, likely tuning the dynamics of developmental gene expression.

Abstract IA10: Regulating transcription elongation at stimulus responsive genes

The majority of mammalian protein-coding genes display transcription in both sense and anti-sense directions. At such bidirectional promoters, Pol II has been shown to initiate transcription and undergo promoter-proximal pausing near both the sense-strand mRNA transcription start site (TSS) and from within the upstream region in the anti-sense direction. This enigmatic, dual promoter structure raises fundamental questions about the specificity, purpose and regulation of upstream anti-sense transcription. Interestingly, transcription in the anti-sense direction is generally non-processive and creates short, unstable non-coding RNAs (ncRNAs). Intriguingly, many distal enhancer regions display bidirectional transcription and generate short unstable ncRNAs, raising parallels between enhancers and anti-sense promoters near protein-coding genes. Importantly, enhancers and upstream anti-sense promoters show exquisite cell type specificity in their location and activity, suggesting key roles in establishing cell state and behavior. Further, perturbation of either transcription or chromatin structure at such non-coding loci has been implicated in the development of cancer and disease, with a profound impact on maintenance of cell state and responsiveness to stimuli. These recent findings raise a number of intriguing questions about the communication between promoters and non-coding loci. Our work has recently defined a mechanism through which upstream anti-sense transcription impacts stimulus-responsive gene expression. Specifically, we found that the distance between sense and anti-sense TSSs at bidirectional promoters strongly influences activation of pro-inflammatory genes in macrophages. We discovered that anti-sense TSSs organize chromatin structure, creating a region of open chromatin between bidirectional TSSs that is optimal for recruitment of the transcription machinery and signal-dependent transcription factors like NF-kB. Further, we discovered that key inflammatory genes possess enhancer-like histone modifications around the anti-sense TSS, and that this characteristic selectively marks genes that are highly activated upon immune stimulation. Citation Format: Karen Adelman, Benjamin S. Scruggs, Telmo Henriques, Adam Burkholder, David C. Fargo. Regulating transcription elongation at stimulus responsive genes. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA10.