Dsif

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs...

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...

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...

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...

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...

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...

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...

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...

STUDY DESIGN: This study compared hip stress among different types of lumbopelvic fusion based on finite element (FE) analysis. OBJECTIVE: We believe that the number and placement of S2 alar iliac (AI) screws and whether the screws loosen likely influence hip joint stress in the FE model. SUMMARY OF BACKGROUND DATA: Spinopelvic fixation has been shown to increase the risk of progression for hip joint osteoarthritis. The biomechanical mechanism is not well understood...

Promoter proximal pausing of RNA Polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DSIF and the inhibitory 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 directly facilitate Pol II pausing...

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...

Several independent studies in the last few years have suggested that phase separation and biomolecular condensation play a critical role in regulating different transcription steps from initiation to pausing and elongation. However, how components of the transcription machinery translocate among different types of condensates during transcription remains poorly understood. Guo et al have now identified a potential mechanism underlying translocation of the DSIF complex from pausing to elongation condensates during promoter pause release, as reported in this issue of EMBO reports...

BACKGROUND: Celiac disease (CD) is frequently associated with type I diabetes mellitus (T1D), where its diagnosis may be a challenging task. This study aims to test the usefulness of the double staining immunofluorescence (dsIF) technique for the detection of intestinal anti-tissue transglutaminase specific IgA antibody (tTG-IgA) deposits in CD and T1D children with coexisting CD. METHODS: A total of 46 patients (30 cases of CD and 16 cases of T1D with CD) and 16 non-diabetic, non-celiac children were recruited...

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)...

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...

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)...

The DSIF complex comprising the Supt4h and Supt5h transcription elongation proteins clamps RNA polymerase II (RNAPII) onto DNA templates, facilitating polymerase processivity. Lowering DSIF components can differentially decrease expression of alleles containing nucleotide repeat expansions, suggesting that RNAPII transit through repeat expansions is dependent on DSIF functions. To globally identify sequence features that affect dependence of the polymerase on DSIF in human cells, we used ultra-deep ChIP-seq analysis and RNA-seq to investigate and quantify the genome-wide effects of Supt4h loss on template occupancy and transcript production...

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...

During gene transcription, RNA polymerase II (RNA Pol II) passes nucleosomes with the help of various elongation factors. Here, we show that RNA Pol II achieves efficient nucleosome passage when the human elongation factors DSIF, PAF1 complex (PAF), RTF1, SPT6, and TFIIS are present. The cryo-EM structure of an intermediate of the nucleosome passage shows a partially unraveled hexasome that lacks the proximal H2A-H2B dimer and interacts with the RNA Pol II jaw, DSIF, and the CTR9trestle helix. RNA Pol II adopts a backtracked state with the RNA 3' end dislodged from the active site and bound in the RNA Pol II pore...

Telomerase RNA has long been considered to be a noncoding component of telomerase. However, the expression of the telomerase RNA gene is not always associated with telomerase activity. The existence of distinct TERC gene expression products possessing different functions were demonstrated recently. During biogenesis, hTR is processed by distinct pathways and localized in different cell compartments, depending on whether it functions as a telomerase complex component or facilitates antistress activities as a noncoding RNA, in which case it is either processed in the mitochondria or translated...