RNA polymerase II pausing in development: orchestrating transcription

The coordinated regulation of transcriptional networks underpins cellular identity and developmental progression. RNA polymerase II promoter-proximal pausing (Pol II pausing) is a prevalent mechanism by which cells can control and synchronize transcription. Pol II pausing regulates the productive elongation step of transcription at key genes downstream of a variety of signalling pathways, such as FGF and Nodal. Recent advances in our understanding of the Pol II pausing machinery and its role in transcription call for an assessment of these findings within the context of development. In this review, we discuss our current understanding of the molecular basis of Pol II pausing and its function during organismal development. By critically assessing the tools used to study this process we conclude that combining recently developed genomics approaches with refined perturbation systems has the potential to expand our understanding of Pol II pausing mechanistically and functionally in the context of development and beyond.

SETD5 Modulates Hematopoietic Stem Cells Homeostasis By Mediating RNA Polymerase II Pausing in Cooperation with HCF-1

Stem cells maintain their self-renewal and differentiation through tight regulation of gene expression patterns at the transcriptional and epigenetic levels. SETD5 is a member of SET domain-containing histone lysine methyltransferase family. Its mutations were identified as the genetic causes of neurodevelopmental disorders, although SETD5 has been shown to lack methyltransferase activity in several studies. Deletion of Setd5 resulted in embryonic lethality at E10.5-11.5 with reduced cell number of CD41 + early hematopoietic cells in the blood island, while the role of SETD5 in adult stem cell, especially in hematopoietic stem cells (HSC) remains unexplored. In this study, by using Vav-Cre and Mx1-Cre mediated conditional knockout murine models, we explored the role of Setd5 in hematopoiesis. We found that Setd5 deficiency led to an enhanced accumulation of HSC in both Setd5 CKO (Vav-Cre; Setd5 fl/fl) and Setd5 IKO (Mx1-Cre; Setd5 fl/fl with pIpC treatment) mice. Cell cycle analysis revealed that higher proportions of SLAM-HSC and LSK + cells underwent active cycling in both Setd5 CKO and Setd5 IKO mice. However, limiting dilution assay revealed a significant ~4-fold decrease of functional HSCs in Setd5 CKO mice, while competitive serial transplantation assays exhibited a progressive decrease in repopulation capacity in Setd5 CKO than that of Setd5 fl/fl. While a progressive decrease in PB chimerism was observed in Setd5 IKO recipients via competitive transplantation assay, myeloid lineage reconstitution was increased, implicating a differentiation bias towards myeloid lineage at the expense of lymphoid lineage of Setd5 IKO HSCs. Thus, our phenotypical studies revealed that Setd5 deficiency impaired the homeostasis of the numbers and function of HSCs. To dissect the underlying mechanism, single cell transcriptome was performed in Setd5 CKO and Setd5 fl/fl control LSK +s using Smart-seq2 method. LSK +s were grouped into 5 clusters with distinct transcriptional features. Significantly, LT stem-like cluster revealed a disruption of LT-HSC and quiescence signatures in Setd5 CKO group, accompanied with an elevated S/G2/M cell cycle signature. When these cells were further projected onto Nestorowa's data (Nestorowa et al, Blood, 2016), Setd5 CKO LSK +s were found to be largely deviated from the core HSC territory toward more differentiated progenitor state. Consistent results were also observed in the bulk RNA-seq of Setd5 CKO and Setd5 IKO SLAM-HSCs. These data indicated that both transcriptional- and immunophenotype-defined LT-HSCs lost the long-term stem cell signatures due to Setd5 deficiency. Although SETD5 has a SET domain, no obvious changes were observed in a majority of histone methylation in Setd5 KO hematopoietic cells. Co-IP assay revealed that SETD5 could interact with HDAC3, PAF1 and HCF-1 complex. SETD5 ChIP-seq revealed that SETD5 could bind to E2F-responsive promoters and regulated the transcription of E2F targets. HCF-1 was reported to interact with E2F and regulate cell cycle entry. HCF-1 and PAF1 was recently found to associate with multiple transcription initiation and elongation complexes to regulate Pol II pausing. We thus investigated whether SETD5 regulates E2F targets via its association with HCF-1, and whether it also regulates E2F targets transcription via modulating Pol II pausing. By analyzing the genome-wide distribution of Pol II in control and Setd5 CKO c-Kit + cells by ChIP-Seq, we found an increase of Pol II occupancy upon SETD5 deficiency. Deletion of Setd5 lead to a significant decrease of pausing index on average which represent a transcription elongation state. Inhibition of the transition of paused Pol II in Setd5 CKO c-Kit + cells with BAY 1143572 (p-TEFb/CDK9 inhibitor) or JQ1 (Brd4 inhibitor) significantly suppressed S phases entry in Setd5 CKO LSK + cells, indicating that increased transition of paused Pol II to elongation mediated by Setd5 depletion could be a main cause for the exit of quiescence stage of Setd5 CKO HSC cells. In summary, we here demonstrate a critical role of Setd5 in HSC maintenance and provide a new insight into the mechanism of Setd5 in regulating HSC quiescence by pausing Pol II-mediated differential expression of cell cycle genes via HCF-1-SETD5-PAF1-Pol II axis. MK.L and YJ.C contributed equally to this work. Corresponding authors: WP.Y and YJ.C. Disclosures: No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Targeting the Transcriptome Through Globally Acting Components

Transcription is a step in gene expression that defines the identity of cells and its dysregulation is associated with diseases. With advancing technologies revealing molecular underpinnings of the cell with ever-higher precision, our ability to view the transcriptomes may have surpassed our knowledge of the principles behind their organization. The human RNA polymerase II (Pol II) machinery comprises thousands of components that, in conjunction with epigenetic and other mechanisms, drive specialized programs of development, differentiation, and responses to the environment. Parts of these programs are repurposed in oncogenic transformation. Targeting of cancers is commonly done by inhibiting general or broadly acting components of the cellular machinery. The critical unanswered question is how globally acting or general factors exert cell type specific effects on transcription. One solution, which is discussed here, may be among the events that take place at genes during early Pol II transcription elongation. This essay turns the spotlight on the well-known phenomenon of promoter-proximal Pol II pausing as a step that separates signals that establish pausing genome-wide from those that release the paused Pol II into the gene. Concepts generated in this rapidly developing field will enhance our understanding of basic principles behind transcriptome organization and hopefully translate into better therapies at the bedside.

Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

A machine learning-based framework for modeling transcription elongation

RNA polymerase II (Pol II) generally pauses at certain positions along gene bodies, thereby interrupting the transcription elongation process, which is often coupled with various important biological functions, such as precursor mRNA splicing and gene expression regulation. Characterizing the transcriptional elongation dynamics can thus help us understand many essential biological processes in eukaryotic cells. However, experimentally measuring Pol II elongation rates is generally time and resource consuming. We developed PEPMAN (polymerase II elongation pausing modeling through attention-based deep neural network), a deep learning-based model that accurately predicts Pol II pausing sites based on the native elongating transcript sequencing (NET-seq) data. Through fully taking advantage of the attention mechanism, PEPMAN is able to decipher important sequence features underlying Pol II pausing. More importantly, we demonstrated that the analyses of the PEPMAN-predicted results around various types of alternative splicing sites can provide useful clues into understanding the cotranscriptional splicing events. In addition, associating the PEPMAN prediction results with different epigenetic features can help reveal important factors related to the transcription elongation process. All these results demonstrated that PEPMAN can provide a useful and effective tool for modeling transcription elongation and understanding the related biological factors from available high-throughput sequencing data.

The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes

For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). The complete NELF complex is formed at the 20E-dependent promoters and participates in both their induced transcriptional response and maintenance of the uninduced state to keep them ready for the forthcoming transcription. NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, we show the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex.

Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. We found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a “no-eye” phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wgRNAi, shows a significant rescue of a reduced eye or a “no-eye” phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. We found that upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. Our data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development.

JMJD5 couples with CDK9 to release the paused RNA polymerase II

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

O-GlcNAc Transferase Activity is Essential for RNA Pol II Pausing in a Human Cell-Free Transcription System

RNA polymerase II pausing is the major regulatory point in transcription in higher eukaryotes. Despite considerable knowledge of the general transcriptional machinery that are required to recruit RNA pol II to a promoter, much less is known how a paused RNA pol II is established and its release regulated, and the entirety of the machinery is likely not known. In part, this is due to the absence of an appropriate biochemical system that functionally recapitulates RNA pol II pausing and elongation and with which the pausing machinery can be identified. We describe herein a cell-free system (CFS) derived from HeLa cells that recapitulates pausing and elongation events known to occur in vivo. We have used this system to show that O-GlcNAc transferase (OGT) activity is required to establish a paused pol II, without which RNA pol II does not pause and instead enters productive elongation. Coupled with previous observations we show that both O-GlcNAc addition and removal are functionally required for pausing and elongation, respectively. Furthermore, the CFS offers significant inroads into understanding RNA pol II pausing and its regulation.