The chromatin remodeler Ino80 mediates RNAPII pausing site determination

Background Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. Results Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. Conclusions Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1

Ewing sarcoma breakpoint region 1 (EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5′ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5′ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5′ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to either early transcription termination, or breakpoint formation.

Linking meta-omics to the kinetics of denitrification intermediates reveals pH-dependent causes of N2O emissions and nitrite accumulation in soil

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N2O reduction despite early transcription of nosZ (mainly clade I), encoding N2O reductase, by diverse denitrifiers. This shows that post-transcriptionally hampered maturation of the NosZ apo-protein at low pH is a generic phenomenon. Identification of transcript reads of several accessory genes in the nos cluster indicated that enzymes for NosZ maturation were present across a range of organisms, eliminating their absence as an explanation for the failure to produce a functional enzyme. nir transcript abundances (for NO2− reductase) in SoilA suggest that low NO2− concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to biological activity. The accumulation of NO2− in neutral soil was ascribed to high nar expression (nitrate reductase). The -omics results revealed dominance of nirK over nirS in both soils while qPCR showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK pool. qnor encoding NO reductase was strongly expressed in SoilA, implying an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2− accumulating soil, was estimated to be ten times higher from SoilA than from SoilN. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

Structural basis of the complete poxvirus transcription initiation process

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I likely fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

The chromatin remodeler Ino80 mediates alternative RNAPII pausing site determination

Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step in early transcription elongation for the precise regulation of gene expression. Here, we provide evidence of promoter-proximal pausing-like distributions of RNAPII in S. cerevisiae. We found that genes bearing an alternative pausing site utilize Ino80p to properly localize RNAPII pausing at the first pausing site and to suppress the accumulation of RNAPII at the second pausing site, which is tightly associated with the +1 nucleosome. This alternative pausing site determination was dependent on the remodeling activity of Ino80p to modulate the +1 nucleosome position and might be controlled synergistically with Spt4p. Furthermore, we observed similar Ino80-dependent RNAPII pausing in mouse embryonic stem cells (mESCs). Based on our collective results, we hypothesize that the chromatin remodeler Ino80 plays a highly conserved role in regulating early RNAPII elongation to establish intact pausing.

Differential SP1 Interactions in SV40 Chromatin from Virions and Minichromosomes

ABSTRACTSP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal by a hit and run mechanism.

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1 via an R-loop-dependent mechanism

SummaryEwing sarcoma breakpoint region 1 (EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5’ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5’ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5’ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to early transcription termination and breakpoint formation.

Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important to maintain the transcriptional activity of Pol II as initiating complexes transition into elongation complexes.