Editorial Overview: RNA polymerase II transcription

RNA polymerase II transcription is a fascinating area of research, with direct relevance to human health and biotechnology. This special issue was organized around an ASBMB meeting entitled “Transcription Regulation: Chromatin and RNA polymerase II” that was scheduled for October, 2020. The plan was to have speakers at the ASBMB meeting contribute to this special issue. Of course, the in-person meeting was canceled because of the pandemic; however, the special issue continued as planned. Included in this issue are nine reviews and three original research articles that encompass areas that are fundamentally important for regulation of RNA polymerase II activity but that remain enigmatic, with many open questions remaining to be addressed.

Because genomic DNA is packaged into chromatin (i.e. DNA wrapped around histones; nucleosomal DNA), transcription in eukaryotic cells requires manipulation of nucleosome-DNA contacts. This is accomplished by chromatin remodeling enzymes, which represent a diverse set of proteins and protein complexes. The structure and function of these remodelers is reviewed by Reyes et al.,[1] which focuses on four major families of remodelers: SWI/SNF, ISWI, CHD, and INO80. A review article from the Narlikar lab[2] provides an in-depth perspective about how chromatin remodelers and RNA polymerase II itself coordinately function at all stages of transcription (initiation, elongation, termination). A notable aspect of this review is the consideration of RNA polymerase II itself as a nucleotide-dependent chromatin remodeler that collaborates with ATP-dependent remodelers to promote gene expression and maintain chromatin structure.

Perhaps the most important class of proteins that control RNA polymerase II activity, genome-wide, is sequence-specific DNA-binding transcription factors (TFs). Among other key functions, TFs help direct the assembly of RNA polymerase II and the so-called general transcription factors (GTFs) at transcription start sites on the genome. The review article by Chen & Pugh[3] describes how TFs act through genomic sequences to control promoter access for GTFs and RNA polymerase II. An interesting observation is that many yeast promoters appear to lack TF requirements for constitutive expression; this model is supported by recent work from the Pugh lab.[4]

A large protein assembly called the pre-initiation complex (PIC) occupies RNA polymerase II transcription start sites in eukaryotic cells, and controls polymerase activity at promoters.[5, 6] After transcription initiation, RNA polymerase II pauses after transcribing about 20–60 nucleotides. This “promoter-proximal pause” is a common regulatory intermediate in metazoans. Review articles by Tim-Michael Decker and Dollinger & Gilmour focus on this important topic. The Decker review[7] provides an in-depth structural and functional analysis of the 2-subunit DSIF complex, which controls not only promoter-proximal pausing, but also transcription elongation. Additional areas covered involve DSIF roles in gene- and enhancer-specific transcription and potential mechanisms for transcription through nucleosomes. The review by Dollinger & Gilmour[8] summarizes the diverse array of factors that govern promoter-proximal pausing and highlights open questions and controversies on this topic. The techniques used to study promoter-proximal pausing are also discussed.

Following promoter-proximal pause release, RNA polymerase II enters an “elongation stage” which generally encompasses gene bodies (i.e. sequences not including the promoter or the transcription termination site). In mammals, gene bodies can be dozens to a few hundred kilobases in length, and so maintaining stable polymerase translocation on the DNA template is critical. RNA polymerase II must also efficiently and rapidly navigate through chromatin during elongation, and the nascent RNA transcript must be managed by RNA processing factors. Elongation is controlled by a set of proteins that are largely distinct from those at promoter-proximal regions. The review by Francette et al.[9] focuses on an essential elongation factor, the multi-subunit Polymerase Associated Factor 1 Complex (PAF1C). Functions of PAF1C that are described include not only RNA polymerase II elongation through chromatin, but also how PAF1C influences epigenetic modifications and nuclear processes not directly related to transcription, such as genome stability, telomere maintenance, and DNA replication. The review by Schärfen & Neugebauer[10] covers a different aspect of RNA polymerase II transcription elongation: the role of the nascent transcript. The nascent transcript, and its potential to form distinct secondary structures, is gaining recognition as a regulator of co-transcriptional RNA processing as well as RNA polymerase II function itself. One reason for this is simply an improved understanding of nascent RNA folding, enabled by novel experimental approaches that probe co-transcriptional RNA structures. These novel methods are also discussed in the review by Schärfen & Neugebauer.

The RNA polymerase II enzyme is different from RNA polymerase I and RNA polymerase III in that it has a long, disordered “C-terminal domain” (CTD) on its large subunit, RPB1 (POLR2A gene in humans). Throughout all stages of transcription (pre-initiation, initiation, elongation, termination), the CTD is a key regulator of RNA polymerase II function. The RNA polymerase II CTD is reviewed in detail by Venkat Ramani et al.[11] Beyond its basic roles in governing transcription and RNA processing, CTD-dependent roles in coordinating histone modifications and chromatin structure are discussed.

One of the next frontiers in the transcription field involves fluorescence imaging technologies, which allow visualization of RNA polymerase II transcription in cells. The final review in this issue focuses on this exciting and fast-moving research area. A noteworthy “deliverable” of single cell imaging experiments is to visualize the location and dynamics of transcription components in the nucleus. These typically manifest experimentally as fluorescent “spots” and this review from Patange et al.[12] critically assesses the capabilities and caveats of current state-of-the-art methods, while also providing a historical perspective. This article should be informative for both novices and experienced researchers working in this area.

Three research articles are included in this special issue, which build upon the topics covered in the reviews. The Musselman lab[13] combined biochemical, mutagenesis, and biophysical experiments toward the BRG1/BRM associated factor (BAF) chromatin remodeling complex (SWI/SNF family). They characterized three conserved domains within the BRM ATPase subunit of the BAF complex that contribute to DNA binding and coordinate functional coupling with the bromodomain. The Liu lab[14] studied the Pontin protein, an AAA+ ATPase linked to diverse nuclear functions. Using biotin ChIP-seq, GRO-seq, proteomics, and other methods, Pontin was identified as a cofactor for the TF RARa; Pontin was shown to help control HOX gene expression during retinoic acid-induced differentiation in pluripotent cells. Finally, the research article from Galburt and co-workers[15] involved a mechanistic analysis of the 10-subunit TFIIH complex, which is generally required for RNA polymerase II to initiate transcription. A combination of biochemical and biophysical assays showed that altered transcription start site usage in yeast (S. cerevisiae) was regulated by a TFIIH-associated kinase module and resulted from differential enzymatic activity of its TFIIH-associated DNA translocase (Ssl2) compared with the human ortholog (XPB).

Collectively, the 12 articles in this special issue provide an excellent overview of the many facets of RNA polymerase II transcription. The nine review articles summarize current understanding in many key areas, while also highlighting unanswered questions and controversies. The three research articles demonstrate how cutting-edge methods can be applied to gain new insights about complex molecular machines (BAF or TFIIH) or to discover new TF-cofactor dependencies. I am grateful for the efforts of the authors and the anonymous reviewers that allowed this special issue to come together, despite the challenging circumstances of the past year. The pandemic has sidelined all in-person scientific conferences, and we have all paid a price from the decreased interactions and conversations. It is encouraging that basic biomedical science has proven “essential” for development of the vaccines,[16] and it appears that these vaccines will allow us to meet in person once again to share new results and talk science. The next ASBMB meeting devoted to “Transcription Regulation: Chromatin and RNA polymerase II” has been scheduled for September 29 - October 2, 2022.