Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Lowering mutant huntingtin by small molecules relieves Huntington's disease symptoms and progression.

Huntington's disease (HD) is an incurable inherited disorder caused by a repeated expansion of glutamines in the huntingtin gene (Htt). The mutant protein causes neuronal degeneration leading to severe motor and psychological symptoms. Selective downregulation of the mutant Htt gene expression is considered the most promising therapeutic approach for HD. We report the identification of small molecule inhibitors of Spt5-Pol II, SPI-24 and SPI-77, which selectively lower mutant Htt mRNA and protein levels in HD cells. In the BACHD mouse model, their direct delivery to the striatum diminished mutant Htt levels, ameliorated mitochondrial dysfunction, restored BDNF expression, and improved motor and anxiety-like phenotypes. Pharmacokinetic studies revealed that these SPIs pass the blood-brain-barrier. Prolonged subcutaneous injection or oral administration to early-stage mice significantly delayed disease deterioration. SPI-24 long-term treatment had no side effects or global changes in gene expression. Thus, lowering mutant Htt levels by small molecules can be an effective therapeutic strategy for HD.

Distinct negative elongation factor conformations regulate RNA polymerase II promoter-proximal pausing.

Metazoan gene expression regulation involves pausing of RNA polymerase (Pol II) in the promoter-proximal region of genes and is stabilized by DSIF and NELF. Upon depletion of elongation factors, NELF appears to accompany elongating Pol II past pause sites; however, prior work indicates that NELF prevents Pol II elongation. Here, we report cryoelectron microscopy structures of Pol II-DSIF-NELF complexes with NELF in two distinct conformations corresponding to paused and poised states. The paused NELF state supports Pol II stalling, whereas the poised NELF state enables transcription elongation as it does not support a tilted RNA-DNA hybrid. Further, the poised NELF state can accommodate TFIIS binding to Pol II, allowing for Pol II reactivation at paused or backtracking sites. Finally, we observe that the NELF-A tentacle interacts with the RPB2 protrusion and is necessary for pausing. Our results define how NELF can support pausing, reactivation, and elongation by Pol II.

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.

DNA-directed termination of RNA polymerase II transcription.

RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo" Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 5' RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end transcription.

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 in vitro 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 in vivo. 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.

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.

RNA Polymerase II "Pause" Prepares Promoters for Upcoming Transcription during Drosophila Development.

According to previous studies, during Drosophila embryogenesis, the recruitment of RNA polymerase II precedes active gene transcription. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. In addition, the composition of the RNA polymerase II "paused" complexes associated with promoters at different developmental stages are described in detail. For this purpose, we performed ChIP-Seq analysis using antibodies for various modifications of RNA polymerase II (total, Pol II CTD Ser5P, and Pol II CTD Ser2P) as well as for subunits of the NELF, DSIF, and PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that during metamorphosis, similar to mid-embryogenesis, the promoters were bound by RNA polymerase II in the "paused" state, preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in a "pause" state was phosphorylated at Ser5 and Ser2 of Pol II CTD and bound the NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex included Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex was phosphorylated at Ser5 of Pol II CTD, but not at Ser2. These results indicate that, during mid-embryogenesis, RNA polymerase II stalls in the "post-pause" state, being phosphorylated at Ser2 of Pol II CTD (after the stage of p-TEFb action). During metamorphosis, the "pause" mechanism is closer to classical promoter-proximal pausing and is characterized by a low level of Pol II CTD Ser2P.

Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes.

Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.

The pleiotropic roles of SPT5 in transcription.

Initially discovered by genetic screens in budding yeast, SPT5 and its partner SPT4 form a stable complex known as DSIF in metazoa, which plays pleiotropic roles in multiple steps of transcription. SPT5 is the most conserved transcription elongation factor, being found in all three domains of life; however, its structure has evolved to include new domains and associated posttranslational modifications. These gained features have expanded transcriptional functions of SPT5, likely to meet the demand for increasingly complex regulation of transcription in higher organisms. This review discusses the pleiotropic roles of SPT5 in transcription, including RNA polymerase II (Pol II) stabilization, enhancer activation, Pol II pausing and its release, elongation, and termination, with a focus on the most recent progress of SPT5 functions in regulating metazoan transcription.

Regulation of Promoter Proximal Pausing of RNA Polymerase II in Metazoans.

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II's subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

Acute stress drives global repression through two independent RNA polymerase II stalling events in Saccharomyces.

In multicellular eukaryotes, RNA polymerase (Pol) II pauses transcription ~30-50 bp after initiation. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now report that nearly all yeast genes, including constitutive and inducible genes, manifest two distinct transcriptional stall sites that are brought on by acute environmental signaling (e.g., peroxide stress). Pol II first stalls at the pre-initiation stage before promoter clearance, but after DNA melting and factor acquisition, and may involve inhibited dephosphorylation. The second stall occurs at the +2 nucleosome. It acquires most, but not all, elongation factor interactions. Its regulation may include Bur1/Spt4/5. Our results suggest that a double Pol II stall is a mechanism to downregulate essentially all genes in concert.

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.

Peptidyl inhibition of Spt4-Spt5: Protein-protein inhibitors for targeting the transcriptional pathway related to C9orf72 expansion repeats.

Spt4/Spt5 is an useful target as it is likely a transcription factor that has implications for long non-coding RNA repeats related to frontotemporal dementia (FTD) found in the C9orf72 disease pathology. Inhibitors for Spt4/Spt5 using peptides as a starting point for assays as a means for developing small molecules, which could likely lead to therapeutic development for inhibition for Spt4/Spt5 with CNS characteristics. To elucidate the specific steps of identification and modification of key interacting residues from Spt4/Spt5 complex with further effect prediction, a set of different computational methods was applied. Newly characterized, theoretically derived peptides docked on Spt4/Spt5 models, based on X-ray crystallography sources, allowed us to complete molecular dynamics simulations and docking studies for peptide libraries that give us high confident set of peptides for use to screen for Spt4/Spt5 inhibition. Several peptides with increased specificity to the Spt4/Spt5 interface were found and can be screened in cell-based assays and enzymatic assays for peptide screens that lead to small molecule campaigns. Spt4/Spt5 comprises an attractive target for neurological diseases, and applying these peptides into a screening campaign will promote the goal of therapeutic searches for FTD drug discovery.

TFIID Enables RNA Polymerase II Promoter-Proximal Pausing.

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.

Structure and mechanism of the RNA polymerase II transcription machinery.

RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.

New Roles for Canonical Transcription Factors in Repeat Expansion Diseases.

The presence of microsatellite repeat expansions within genes is associated with >30 neurological diseases. Of interest, (GGGGCC)>30-repeats within C9orf72 are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). These expansions can be 100s to 1000s of units long. Thus, it is perplexing how RNA-polymerase II (RNAPII) can successfully transcribe them. Recent investigations focusing on GGGGCC-transcription have identified specific, canonical complexes that may promote RNAPII-transcription at these GC-rich microsatellites: the DSIF complex and PAF1C. These complexes may be important for resolving the unique secondary structures formed by GGGGCC-DNA during transcription. Importantly, this process can produce potentially toxic repeat-containing RNA that can encode potentially toxic peptides, impacting neuron function and health. Understanding how transcription of these repeats occurs has implications for therapeutics in multiple diseases.

Targeting Spt5-Pol II by Small-Molecule Inhibitors Uncouples Distinct Activities and Reveals Additional Regulatory Roles.

Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.

In vitro analysis of RNA polymerase II elongation complex dynamics.

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.