DRB Sensitivity-inducing Factor Research Articles

Structural basis of Integrator-dependent RNA polymerase II termination

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator–PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10–INTS13–INTS14–INTS15 module that may use its ‘sting’ to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator–PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

NELF and PAF1C complexes are core transcriptional machineries controlling colon cancer stemness

Mutations in APC, found in 80% of colon caner, enhance β-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional mechanism underlying the induction of colon cancer stemness by stable β-catenin remains unclear. Here, we found that inducible inhibition of β-catenin suppressed elongation of Pol II and RNA polymerase-associated factor 1 complex (PAF1C) around the transcription start site (TSS) of LGR5. Moreover, stable β-catenin enhanced the formation of active Pol II complex cooperatively with CDC73 and CDK9 by facilitating the recruitment of DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) complexes to the Pol II complex. Subsequently, stable β-catenin facilitated the formation of the Pol II–DSIF–PAF1C complex, suggesting that stable β-catenin induces cancer stemness by stimulating active Pol II complex through NELF and PAF1C. Furthermore, NELF or PAF1C inhibition recapitulated the changes in cancer stemness-related gene expression induced by the inhibition of stable β-catenin and suppressed colon cancer stemness. Additionally, the chemical inhibition of CDK12 (a downstream transcription CDK of PAF1C) suppressed colon cancer stemness. These results suggest that NELF and PAF1C are the core transcriptional machineries that control expression of colon cancer stemness-inducing genes and may be therapeutic targets for colon cancer.

Emerging Roles of SPT5 in Transcription.

Suppressor of Ty homolog-5 (SPT5) discovered in the yeast mutant screens as a suppressor of mutation caused by the insertion of the Transposons of yeast (Ty) element along with SPT4, with which it forms a holoenzyme complex known as DRB sensitivity-inducing factor (DSIF) and plays an essential role in the regulation of transcription. SPT5 is a highly conserved protein across all three domains of life and performs critical functions in transcription, starting from promoter-proximal pausing to termination. We also highlight the emerging role of SPT5 in other non-canonical functions, such as the regulation of post-translational modifications (PTM) and the transcriptional regulation of non-coding genes. Also, in brief, we highlight the clinical implications of SPT5 dysregulation.

Structural basis of the human negative elongation factor NELF-B/C/E ternary complex

Negative elongation factor (NELF) is a four-subunit transcription elongation factor that mainly functions in maintaining the paused state of RNA polymerase II in eukaryotes. Upon binding to Pol II, NELF works synergistically with DRB sensitivity-inducing factor (DSIF) and inhibits transcription elongation of Pol II, which subsequently retains a stably paused state 20–60 base pairs downstream of the promoter. The promoter-proximal pausing of Pol II caused by NELF is a general mechanism of transcriptional regulation for most signal-responsive genes. To date, structural studies have significantly advanced our understanding of the molecular mechanisms of NELF. However, a high quality structural model clarifying the interaction details of this complex is still lacking. In this study, we solved the high resolution crystal structure of the NELF-B/C/E ternary complex. We observed detailed interactions between subunits and identified residues important for the association between NELF-B and NELF-E. Our work presents a precise model of the NELF complex, which will facilitate our understanding of its in vivo function.

Assessment of the roles of Spt5-nucleic acid contacts in promoter proximal pausing of RNA polymerase II

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DRB sensitivity-inducing factor (DSIF) and the inhibitory negative elongation factor (NELF). DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We used a highly purified invitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. We identified two domains of Spt5, the KOW4 and NGN domains, that facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha-helical motif is also needed to support fly viability. We also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal invivo. Finally, we show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex. In summary, our results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing.

Structural insights into human co-transcriptional capping

Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

Role of RNA Polymerase II Promoter-Proximal Pausing in Viral Transcription.

The promoter-proximal pause induced by the binding of the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) to RNAP II is a key step in the regulation of metazoan gene expression. It helps maintain a permissive chromatin landscape and ensures a quick transcriptional response from stimulus-responsive pathways such as the innate immune response. It is also involved in the biology of several RNA viruses such as the human immunodeficiency virus (HIV), the influenza A virus (IAV) and the hepatitis delta virus (HDV). HIV uses the pause as one of its mechanisms to enter and maintain latency, leading to the creation of viral reservoirs resistant to antiretrovirals. IAV, on the other hand, uses the pause to acquire the capped primers necessary to initiate viral transcription through cap-snatching. Finally, the HDV RNA genome is transcribed directly by RNAP II and requires the small hepatitis delta antigen to displace NELF from the polymerase and overcome the transcriptional block caused by RNAP II promoter-proximal pausing. In this review, we will discuss the RNAP II promoter-proximal pause and the roles it plays in the life cycle of RNA viruses such as HIV, IAV and HDV.

Measuring the precise position of transitions in transcription with PRO‐IP‐seq

Transcription is a fundamental process for all known organisms. Transcription by RNA Polymerase II (Pol II), the enzyme responsible for mRNA synthesis, is highly regulated by protein factors at every step. In multicellular eukaryotes, Pol II pauses 20‐60 bases downstream of the transcription start site in nearly all genes. This promoter‐proximal pausing is established as an important step in transcription regulation and is dependent on several pausing and elongation factors. The protein complexes DSIF (DRB Sensitivity Inducing Factor) and NELF (Negative Elongation Factor) bind to Pol II early in transcription to induce pausing. Pause release occurs upon phosphorylation of Pol II, DSIF and NELF by the Cdk9, Cyclin T1 heterodimer P‐TEFb (Positive Transcription Elongation Factor b). Although Pol II pausing is a key step in transcription regulation and has been extensively studied, the molecular mechanisms of pause and pause release are incompletely understood. Furthermore, recent studies have implicated controlled termination early in the gene as a mechanism to control transcription. A challenge to studying these key regulatory steps is that they occur in a narrow window: from initiation to Pol II pausing and release occurs in 20‐60 base range. Current approaches to study transcription factor interactions are unable to provide high enough resolution or sensitivity to address precisely where key transitions in transcription occur.To address this problem, I am developing an assay to measure the exact position of Pol II, when it is bound by a factor of interest. The technique couples our lab’s Precision Run‐on sequencing technique (PRO‐seq), which measures the position of active Pol II with single base‐pair resolution, and an immunoprecipitation enrichment step. Initial PRO‐IP‐seq (Precision Run‐on Immunoprecipitation sequencing) data shows that we can enrich for populations of Pol II bound by specific transcription factors. Utilizing this tool, we will be able to measure genomically where both stable and more transient transcription factors interact with Pol II at near single‐base resolution.

Investigating the Effect of PARP Mediated Compartments on Transcription Dynamics

Transcription of DNA into RNA is a fundamental, universal, and highly regulated process in all living organisms. RNA Polymerase II (Pol II), is the enzyme that catalyzes this reaction and it is highly regulated by the pausing factors DRB Sensitivity Inducing Factor (DSIF) and Negative Elongation Factor (NELF), and elongation factors, including Positive Transcription Elongation Factor b (P‐TEFb) and Polymerase Associated Factor 1 (PAF1). However, most previous studies of these factors have only provided snapshots of factor binding or snapshots of mRNA production following their depletion. While valuable, these studies provide limited information about the time frames in which Pol II and key transcription factors interact with each other and with the DNA. Using live‐cell imaging of amplified and interphase‐extended Drosophila polytene chromosomes with endogenously tagged transcription factors and Pol II, we can analyze the real time behavior of these molecules at single genetic loci and at high resolution. In particular, heat shock genes, like heat shock protein 70 (Hsp70), have been shown to be a robust system for studying transcription because of their inducible nature; they are able to transition quickly from a paused to a highly active state. Previous work from our lab utilizing this system suggested the formation of a transcription compartment during prolonged bouts of transcription. We propose that transcription compartments form around Hsp70 loci during an extended heat shock due to the activity of Poly ADP Ribose Polymerase (PARP) polymerizing large PAR chains that trap Pol II and other transcription factors, restricting their movement and preventing free diffusion with other molecules in the nucleus. We are tagging proteins of interest with photo‐activatable fluorescent proteins to minimize background by selectively activating a population of molecules only in the small 3‐D volume of specific loci. Utilizing this system we have begun measuring the dynamics of Pol II at specific loci and are working toward measuring the dynamics of key transcription regulating factors and further characterizing PARP‐mediated transcription compartments.

Targeting CDK9 for Anti-Cancer Therapeutics.

Simple SummaryCDK9, in combination with Cyclin T1, is one of the major regulators of RNA Polymerase II mediated productive transcription of critical genes in any cell. The activity of CDK9 is significantly up-regulated in a wide variety of cancer entities, to aid in the overexpression of genes responsible for the regulation of functions, which are beneficial to the cancer cells, like proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Enhanced CDK9 activity, therefore, leads to poorer prognosis in many cancer types, offering the rationale to target it using small-molecule inhibitors. Several, increasingly specific inhibitors, have been developed, some of which are presently in clinical trials. Other approaches being tested involve combining inhibitors against CDK9 activity with those against CDK9’s upstream regulators like BRD4, SEC and HSP90; or downstream effectors like cMYC and MCL-1. The inhibition of CDK9’s activity holds the potential to be a highly effective anti-cancer therapeutic.Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90.

CRISPRi-mediated depletion of Spt4 and Spt5 reveals a role for DSIF in the control of HIV latency

Pivotal studies on the control of HIV transcription has laid the foundations for our understanding of how metazoan transcription is executed, and what are the factors that control this step. Part of this work established a role for DRB Sensitivity Inducing Factor (DSIF), consisting of Spt4 and Spt5, in promoting pause-release of RNA Polymerase II (Pol II) for optimal elongation. However, while there has been substantial progress in understanding the role of DSIF in mediating HIV gene transcription, its involvement in establishing viral latency has not been explored. Moreover, the effects of depleting Spt4 or Spt5, or simultaneously knocking down both subunits of DSIF have not been examined. In this study, we employed CRISPR interference (CRIPSRi) to knockdown the expression of Spt4, Spt5 or the entire DSIF complex, and monitored effects on HIV transcription and viral latency. Knocking down DSIF, or each of its subunits, inhibited HIV transcription, primarily at the step of Tat transactivation. This was accompanied by a decrease in promoter occupancy of Pol II and Cdk9, and to a lesser extent, AFF4. Interestingly, targeting the expression of one subunit of DSIF, reduced the protein stability of its counterpart partner. Moreover, depletion of Spt4, Spt5 or DSIF complex impaired cell growth, but did not cause cell death. Finally, knockdown of Spt4, Spt5 or DSIF, facilitated entry of HIV into latency. We conclude that each DSIF subunit plays a role in maintaining the stability of its other partner, achieving optimal function of the DSIF to enhance viral gene transcription.

Dynamic regulation of promoter-proximal RNA polymerase II pausing

Transcriptional pausing is a widely used regulatory mechanism to control precise gene expression from bacteria to humans. In metazoans, transcription initiation requires the recruitment of general transcription factors and RNA polymerase II (Pol II) to gene promoters. After it initiates transcription and synthesizes a short RNA, Pol II pauses at the promoter-proximal region, standing by for further cues to enter the productive elongation stage. Promoter-proximal Pol II pausing is one of the rate-limiting steps during transcription, functioning as an early transcriptional elongation check point, to ensure the correct modification of transcription machinery and the addition of the 7-methylguanosine cap to nascent transcripts. Pol II pausing at promoter-proximal region is stabilized by the negative elongation factor (NELF) and the 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). In response to developmental signals or environmental stress, the release of paused Pol II can be triggered by the positive transcription elongation factor b (P-TEFb), which is composed of the kinase CDK9 and its regulatory subunit CCNT1/2. The kinase activity of P-TEFb towards Pol II carboxy-terminal domain (CTD), NELF and DSIF is critical for P-TEFb to execute its function in pause release, and is thus tightly controlled. To date, it has been identified that P-TEFb mainly exists in three different complexes: the super elongation complex (SEC), BRD4-P-TEFb, and the inactive 7SK snRNP-P-TEFb complex. SEC, one of the most active P-TEFb containing complexes, is required for rapid gene expression in response to stress or developmental signals. The active and inactive forms of P-TEFb complexes co-exist in vivo , reaching equilibrium to meet the transcriptional needs of cells. The transition of Pol II from pause to productive elongation stage is achieved by dynamic exchange between the pausing factors and the positive elongation factors at the proximal promoter region. Liquid-liquid phase separation (LLPS) in living cells is a physicochemical process that confines biochemical reactions within membrane-less compartments. Low complexity intrinsically disordered regions (IDRs) within proteins can initiate and promote phase separation and formation of liquid droplets, which in turn facilitate the multivalent weak interactions among these IDR-containing proteins. Recent studies using super-resolution live cell imaging technology have revealed that RNA Pol II can form dynamic foci in cells through LLPS and transitions between different phases, which are likely mediated by phosphorylated CTD. Moreover, factors involved in various transcription steps can rapidly and dynamically concentrate and exchange at the hot regulatory genomic loci. For example, SEC components are able to compartmentalize and concentrate P-TEFb from its inactive partners. Upon serum stimulation, SEC assembles promptly into phase-separated droplets at the immediate early genes (IEG), triggering rapid transcription induction. Mutations in transcription regulators can lead to developmental defects and many human diseases, including cancers. For instance, abnormal stabilization of SEC components via MLL fusion plays a contributing role in leukemogenesis. Fusion of the SEC core component ENL with MLL can greatly enhance phase separation of SEC, through which the leukemic genes might be mis-activated. In addition, there is growing evidence that disease related mutations in transcriptional regulators can cause malfunction through aberrant condensation, providing potential targets for designing therapeutic strategies through phase separation interventions.

PTEN modulates gene transcription by redistributing genome-wide RNA polymerase II occupancy.

Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30–60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it’s required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.

Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.

Structure of paused transcription complex Pol II-DSIF-NELF.

Metazoan gene regulation often involves pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy (cryo-EM) structure of the paused Sus scrofa/Homo sapiens Pol II-DSIF-NELF transcription elongation complex (PEC) at 3.2 Å resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate (NTP) substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing factor TFIIS. Additionally, NELF possesses two flexible tentacles that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding NELF function during promoter-proximal gene regulation.

Targeting of RNA Polymerase II by a nuclear Legionella pneumophila Dot/Icm effector SnpL.

The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localise to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localises to the nucleus where they subvert host cell transcriptional responses to infection. Here, we identified Lpw27461 (Lpp2587), Lpg2519 as a new nuclear-localised effector that we have termed SnpL. Upon ectopic expression or during L.pneumophila infection, SnpL showed strong nuclear localisation by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, Suppressor of Ty5 (SUPT5H)/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex that regulates RNA Polymerase II dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central Kyprides, Ouzounis, Woese motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L.pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression.

Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing

DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In Drosophila and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depleted Drosophila nuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA.

RNA polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II.

Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.

CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription

CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3’ box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.

Control of Transcriptional Elongation

Elongation is becoming increasingly recognized as a critical step in eukaryotic transcriptional regulation. Although traditional genetic and biochemical studies have identified major players of transcriptional elongation, our understanding of the importance and roles of these factors is evolving rapidly through the recent advances in genome-wide and single-molecule technologies. Here, we focus on how elongation can modulate the transcriptional outcome through the rate-liming step of RNA polymerase II (Pol II) pausing near promoters and how the participating factors were identified. Among the factors we describe are the pausing factors--NELF (negative elongation factor) and DSIF (DRB sensitivity-inducing factor)--and P-TEFb (positive elongation factor b), which is the key player in pause release. We also describe the high-resolution view of Pol II pausing and propose nonexclusive models for how pausing is achieved. We then discuss Pol II elongation through the bodies of genes and the roles of FACT and SPT6, factors that allow Pol II to move through nucleosomes.