Journal of Molecular Biology
Review ArticleRegulation of Promoter Proximal Pausing of RNA Polymerase II in Metazoans
Graphical abstract
Introduction
Eukaryotic transcription is a highly regulated process that requires precise temporal and spatial coordination of a multitude of factors at the initiation, elongation, and termination stages. Promoter proximal pausing of RNA polymerase II (Pol II) has emerged as a significant step in the canonical transcription cycle. In metazoans, transcription initiation is followed by an accumulation of Pol II ~30–60 nt downstream of the transcription start site.1 This step is not present in Saccharomyces cerevisiae and Caenorhabditis elegans, likely owing to the absence of the key negative elongation factor (NELF). The first evidence of metazoan Pol II pausing in vivo emerged when the Chambon lab, using a nuclear run-on assay, observed a concentration of Pol II at the 5′end of the beta-globin gene in nuclei from mature hen erythrocytes that were anticipated to be transcriptionally silent.2 Similar phenomena were later observed on mammalian c-myc,3, 4, 5 HIV-16 and on non-induced Drosophila heat shock genes.7, 8 The study of the Drosophila hsp70 heat shock gene by Gilmour and Lis was particularly groundbreaking. Using protein-DNA crosslinking with UV light, a predecessor to the widely used chromatin immunoprecipitation techniques of today, they determined that a single Pol II molecule associates with the region between −12 and +65 on the non-induced hsp70 gene.7 Subsequent work by Rougvie and Lis determined that the Pol II at the 5′end of genes in Drosophila is transcriptionally engaged8 and subsequent permanganate footprinting analyses in living cells revealed the transcription bubble associated with these Pol II.9 The question remained, however, as to whether the paused Pol II was an idiosyncrasy of a small number of genes or a more general phenomenon. The advent of genomic methods provided resounding support for the latter, leading to the notion that promoter proximal pausing is a ubiquitous step in the transcription cycle for most if not all protein encoding genes in mammals and Drosophila.10, 11, 12, 13 Pausing has been linked to a number of regulatory functions, including developmental control 12, 14, 15, 16and the maintenance of transcriptionally permissive, nucleosome-free regions around promoters.17, 18 Indeed, it is this latter observation that can explain the seemingly contradictory finding that depletion of the pausing factor NELF results in a decrease rather than an increase in expression of some genes.18
Promoter proximal pausing requires at least two factors which function cooperatively: DRB sensitivity-inducing factor (DSIF) and NELF.19, 20, 21, 22 The cyclin dependent kinase positive transcription elongation factor b (P-TEFb) appears to facilitate pause release by phosphorylating Pol II, DSIF, and NELF, resulting in the dissociation of NELF from the elongation complex and the transition of DSIF from a negative elongation factor to a positive elongation factor.21, 23, 24 Extensive genome-wide and biochemical studies have provided a general framework for how pausing occurs, yet many of the underlying mechanisms remain an area of inquiry. First, the exact mechanism through which DSIF and NELF promote pausing, particularly with regards to the role of DSIF, has yet to be fully elucidated. Several factors in addition to NELF and DSIF have been implicated in pausing, most notable among them transcription factor II D (TFIID), GAGA factor, and RNA polymerase II-associated factor 1 complex (PAF1C), yet the exact role of each of these factors and the nature of their interactions with the paused complex requires further study. Finally, additional considerations, such as the role of DNA/RNA sequence, the duration and stability of the pause, and the relationship between Pol II pausing and chromatin architecture remain areas of continuing or emerging research and even debate.
Promoter proximal pausing of Pol II is governed by several intrinsic and extrinsic elements. Increasing evidence points to DNA and RNA sequence playing a role in regulating promoter proximal pausing. Enrichment of promoter elements and pause motifs, as well as GC content, likely influence the stability and efficiency of the pause.25, 26, 27 Experiments in Drosophila show that disrupting downstream promoter elements can shift the location of the pause on the hsp70 gene, suggesting that pause position is heavily reliant on DNA sequence, but it is unclear whether this result can be widely extrapolated for other promoters.28 Indeed, contrasting evidence has shown that the location of the pause depends on the kinetics of the elongating Pol II; lowering nucleotide concentrations or replacing the wild type Pol II with a slowly elongating mutant shifts the location of the pause upstream.29 Thus, the role of intrinsic Pol II-nucleic acid interactions in pausing remains a topic of ongoing controversy.
Extrinsic interactions, such as those between Pol II and DSIF, NELF, and P-TEFb, as well as between Pol II and accessory factors such as TFIID and others play critical roles in establishing the pause and governing the dynamics of pause stability and pause release. Interactions between the nucleic acid scaffold and DSIF and NELF are also likely significant contributors to the pausing mechanism. Moreover, since the discovery of Pol II pausing, the role of chromatin architecture has been a subject of controversy. Of particular interest has been the +1 nucleosome, which may play a significant role in enhancing Pol II pausing in humans,30 but likely affects Drosophila Pol II promoter proximal pausing in a promoter-specific manner.31
Taken together, the current body of knowledge about Pol II promoter proximal pausing suggests a multipartite mechanism that relies on the interplay between intrinsic and extrinsic interactions, as well as on the promoter and chromatin context of the pause. This review distinguishes itself from other recent reviews on Pol II elongation and promoter proximal pausing in several ways.32, 33, 34, 35, 36 First, we provide an overview of the broad range of the tools available for studying promoter proximal pausing, including in vivo, cell free, and genomic techniques. Furthermore, we describe the factors involved in promoter proximal pausing with a particular emphasis on the structural and molecular mechanisms of DSIF and NELF function. We also discuss the emerging evidence that suggests TFIID plays a significant and context-dependent role as a regulator of promoter proximal pausing. Additionally, we discuss and attempt to resolve some of the controversies regarding the role of chromatin architecture in promoter proximal pausing, with an emphasis on the differences between human and Drosophila systems. Finally, we provide a holistic model for Pol II pausing that illustrates the various protein factors implicated in pausing and their interactions and reconciles some of the apparent contradictions in the current body of literature.
Section snippets
Tools to study pausing
In the almost three decades since the discovery of promoter proximal pausing, numerous tools have emerged to facilitate its study (Table 1). Genome-wide techniques like ChIP-seq37 and the higher resolution ChIP-exo38, 39 and ChIP-nexus40, 41 have emerged as powerful tools that can be utilized to determine the location of Pol II and its binding partners. These techniques involve crosslinking proteins to DNA in vivo followed by immunoprecipitation of protein-DNA adducts. ChIP-exo and ChIP-nexus
DSIF
DSIF is a two-subunit transcription factor composed of Spt4 and Spt5 and is widely conserved across eukaryotes.75 In metazoans, DSIF acts to stimulate or repress transcription elongation, depending on its phosphorylation state. The pausing function of DSIF was first discovered by the Handa lab as an activity that rendered Pol II transcription sensitive to inhibition by DRB, a nucleoside analog.19 DSIF associates with the Pol II elongation complex after the transcription of at least eighteen
NELF
NELF was first identified as a four-subunit complex (NELF-A, -B, -C/D isoforms, -E) that works cooperatively with DSIF to repress Pol II elongation.20, 79 NELF is recruited to the elongation complex by DSIF22 and plays a multifaceted role in establishing and maintaining promoter proximal pausing.
Phosphorylation-dependent regulation of the pause
P-TEFb, predominantly composed of Cdk995 and Cyclin T in Drosophila96 and Cdk995 and Cyclin T1 or Cyclin T2 in human cells97 is broadly considered to be critical for the release of Pol II from the pause and its transition into productive elongation. P-TEFb is frequently found as a part of larger complexes. In its inhibited form, P-TEFb is associated with the 7SK noncoding RNA and the MeCPE, LARP7, and HEXIM factors.98 In its most active form, P-TEFb is part of the multi-subunit super-elongation
TFIID
TFIID is a general transcription factor that provides the foundation for assembling a preinitiation complex (PIC). The relationship between TFIID and Pol II promoter proximal pausing remains a topic of some controversy. In 2019, the Biswas lab used co-immunoprecipitation experiments to show that the TAF subunit of TFIID interacts with components of the SEC both in mammalian cells and in vitro. AF9 and EAF1, two SEC subunits, both have serine-rich domains that are required for this interaction.
GAGA factor
GAGA factor (GAF) is a sequence-specific DNA binding factor that has been shown to promote promoter proximal pausing in Drosophila. Early work from the Lis lab indicated that point mutations in the GAGA element significantly decrease pausing on the Drosophila hsp70 promoter.118, 119 However, it was unclear whether the loss of paused Pol II was due to disruption of pausing or to diminished transcription initiation, a prerequisite for pausing. The Gilmour lab later established that the GAGA
PAF1C
The multi-subunit Paf1 complex (PAF1C) has been implicated in promoter proximal pausing but its role is controversial. Initial studies by the Shilatifard group showed that PAF1C loss results in release of paused Pol II into gene bodies in both Drosophila and human HCT116 and MCF7 cell lines, with a greater effect observed at highly paused genes. PAF1 knockdown also results in increased SEC recruitment and Pol II CTD phosphorylation at the Ser2 position.124 Hence, they proposed that PAF1C helps
Additional proteins
Since the discovery of DSIF and NELF, many more proteins have been implicated in promoter proximal pausing of Pol II and it is likely that more will be discovered in the future. We discuss some of them in the following section but note that the complete picture remains to be fully resolved.
Recently, the pre-exon junction complex (pre-EJC), which regulates expression of long genes in Drosophila has also been identified as an intron length-dependent facilitator of Pol II pausing. Knockdown of the
Role of sequence in pausing
Promoter elements such as GAGA, Inverted GAGA, Inr, and DPE are enriched at paused genes in Drosophila.25, 42 Some of these, such as the Inr and DPE elements, are TFIID binding sites and are likely key players in regulating differential TFIID function, as discussed above. In Drosophila, sequence can act as a particularly strong contributor to Pol II pause stability. Reporter-ChIP-nexus experiments, which detect pausing on plasmids transfected into cells, have shown that an Inr variant with a G
Drosophila versus human and the role of the +1 nucleosome
The role of nucleosome organization, in particular the positioning of the +1 nucleosome, in establishing and maintaining Pol II promoter proximal pausing has been the subject of several contradictory studies. Early experiments by the Luse and Kingston labs established that partially purified Pol II elongation complexes from HeLa cells pause on templates containing assembled nucleosomes.134, 135 Mapping of micrococcal nuclease cuts in HeLa nuclei also determined that human hsp70 is nucleosomal
Duration of the pause
The duration of the pause and whether Pol II, upon release, transitions to productive elongation or prematurely terminates transcription impacts the level of gene expression. These dynamics were initially studied by blocking transcription initiation with the chemical triptolide, which covalently inhibits the ATPase activity of the TFIIH subunit XPB.142This ATPase activity plays a critical role in overcoming a block to initiation imposed by XPB.143, 144 The stability of the paused Pol II is then
Open questions and future directions
Promoter proximal pausing of Pol II requires NELF and DSIF and may also involve accessory factors such as TFIID, PAF1, GAF (in Drosophila), the pre-EJC, and FACT, as well as chromatin remodelers and histone modifiers (Figure 5). However, for many of these factors, how they promote pausing in the context of the Pol II-DSIF-NELF complex remains a mystery. Do they associate directly with the elongation complex or do some of them, such as FACT and other chromatin-associated factors, mediate pausing
Accession numbers
The following structures were used to generate the figures in this review: PDB ID: 6GML, structure of human Pol II-NELF-DSIF complex; PDB ID: 6GMH, structure of human Pol II-DSIF-SPT6-PAF1C; PDB ID: 5OIK, structure of Pol II-DSIF; PDB ID: 6ASX, structure of paused bacterial RNA Polymerase; PDB ID: 6ALF, structure of post-translocated bacterial RNA Polymerase.
CRediT authorship contribution statement
Roberta Dollinger: Writing - original draft, Writing - review & editing. David S. Gilmour: Writing - review & editing, Funding acquisition.
Acknowledgements
Funding for this work was provided by NIH grant GM047477 to David S. Gilmour. We thank Seychelle M. Vos for thoughtful reading of the manuscript.
Declarations of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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