Recent years have witnessed a sea change in our understanding of transcription regulation: whereas traditional models focused solely on the events that brought RNA polymerase II (Pol II) to a gene promoter to initiate RNA synthesis, emerging evidence points to the pausing of Pol II during early elongation as a widespread regulatory mechanism in higher eukaryotes. Current data indicate that pausing is particularly enriched at genes in signal-responsive pathways. Here the evidence for pausing of Pol II from recent high-throughput studies will be discussed, as well as the potential interconnected functions of promoter-proximally paused Pol II.

Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

http://compbio.mit.edu/publications/47_Kharchenko_Nature_10.pdf

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster

Recent studies of transcription have revealed a level of complexity not previously appreciated even a few years ago, both in the intricate use of post-initiation control and the mass production of rapidly degraded transcripts. Dissection of these pathways requires strategies for precisely following transcripts as they are being produced. Here we present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3' ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq in Saccharomyces cerevisiae reveals that although promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus, nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.

/pdf/nascent-transcript-sequencing-visualizes-transcription-at-4viitppspt.pdf

Nascent transcript sequencing visualizes transcription at nucleotide resolution

Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.

Getting up to speed with transcription elongation by RNA polymerase II

Emerging evidence indicates that gene expression in higher organisms is regulated by RNA polymerase II stalling during early transcription elongation. To probe the mechanisms responsible for this regulation, we developed methods to isolate and characterize short RNAs derived from stalled RNA polymerase II in Drosophila cells. Significant levels of these short RNAs were generated from more than one-third of all genes, indicating that promoter-proximal stalling is a general feature of early polymerase elongation. Nucleotide composition of the initially transcribed sequence played an important role in promoting transcriptional stalling by rendering polymerase elongation complexes highly susceptible to backtracking and arrest. These results indicate that the intrinsic efficiency of early elongation can greatly affect gene expression.

https://science.sciencemag.org/content/sci/327/5963/335.full.pdf

Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II in Drosophila

Physical activity is regulated by a variety of genetic molecules. However, the pathways through which those molecules work to regulate activity is largely unknown. The purpose of this study was to gather the known genetic molecules that are associated with activity regulation and define overall upstream regulator pathways through which these molecules work. We conducted a systematic review to gather all available published datasets related to physical activity regulation, standardized the data for genomic location and species, and used this data, in an unbiased manner to create a dataset that was used: (1) to physically map and visualize all identified molecules to homologous chromosome locations and (2) as the dataset for which an Upstream Regulator Analysis (URA) was conducted using Qiagen Ingenuity Pathway Analysis (IPA) software. Our search resulted in 469 genetic molecules (e.g. genomic variant, transcript, protein, micro-RNA) that were split into brain (n=366) and muscle (n=345) sub-groups, which was our attempt to separate differences in central vs peripheral pathways. The brain and muscle data sets had several potential upstream regulators, the top-rated being β-estradiol as a regulator for 19.5% and 21% of the brain and muscle datasets respectively. To our knowledge, β-estradiol’s identification as a potential regulator, is the first evidence to link the well-known effects of sex hormones on physical activity with genetic regulation of physical activity. There were a variety of potential upstream regulators for the molecules collected in this review, but interestingly, three of the top five for both brain and muscle are nuclear receptor binding ligands; estradiol (estrogen receptor), dexamethasone (glucocorticoid receptor), and tretinoin (retinoic acid receptor), indicating a potential role of nuclear receptors in the regulation of physical activity. Selective nuclear receptor modulation may be an area of interest in future mechanistic studies of the genetic regulation of physical activity.

/pdf/it-all-rolls-downstream-upstream-control-of-physical-bghbbqz3.pdf

Liwen Liu

Papers

Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II in Drosophila

“It All Rolls Downstream: Upstream Control of Physical Activity Regulation”