Publisher Correction: Promoter-proximal RNA polymerase II termination regulates transcription during human cell type transition
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Promoter-proximal RNA polymerase II termination regulates transcription during human cell type transition
Metazoan gene transcription by RNA polymerase II (Pol II) is regulated in the promoter-proximal region. Pol II can undergo termination in the promoter-proximal region but whether this can contribute to transcription regulation in cells remains unclear. Here we extend our previous multiomics analysis to quantify changes in transcription kinetics during a human cell type transition event. We observe that upregulation of transcription involves an increase in initiation frequency and, at a set of genes, a decrease in promoter-proximal termination. In turn, downregulation of transcription involves a decrease in initiation frequency and an increase in promoter-proximal termination. Thus, promoter-proximal termination of Pol II contributes to the regulation of human gene transcription.
Mechanism of expression regulation of head-to-head overlapping protein-coding genes INO80E and HIRIP3
Although the existence of overlapping protein-coding genes in eukaryotic genomes is known for decades, their role in regulating expression remains far from fully understood. Here, the mechanism regulating the expression of head-to-head overlapping genes, a pair of INO80E and HIRIP3 genes is presented. Based on a series of experiments, we show that the expression of these genes is strongly dependent on sense/antisense interactions. The overlapping transcripts form an RNA:RNA duplex that has a stabilizing effect on the mRNAs involved, and this stabilization may be mediated by the ELAVL1 protein. We also show that the transcription factor RARG is important for the transcription of both genes studied. In addition, we demonstrate that the overlapping isoform of INO80E forms an R-loop that may positively regulate HIRIP3 isoforms. We propose that both structures, dsRNA and R-loops, help to keep the DNA loop open to allow the transcription of the remaining variants of both genes. However, experiments suggest that RNA:RNA duplex formation plays a major role, while R-loops play only a complementary one. The absence of this dsRNA structure leads to the loss of a stable DNA opening and consequently to transcriptional interference.
Structural basis for the activation of plant bunyavirus replication machinery and its dual-targeted inhibition by ribavirin
Despite the discovery of plant viruses as a new class of pathogens over a century ago, the structure of plant virus replication machinery and antiviral pesticide remains lacking. Here we report five cryogenic electron microscopy structures of a ~330-kDa RNA-dependent RNA polymerase (RdRp) from a devastating plant bunyavirus, tomato spotted wilt orthotospovirus (TSWV), including the apo, viral-RNA-bound, base analogue ribavirin-bound and ribavirin-triphosphate-bound states. They reveal that a flexible loop of RdRp’s motif F functions as ‘sensor’ to perceive viral RNA and further acts as an ‘adaptor’ to promote the formation of a complete catalytic centre. A ten-base RNA ‘hook’ structure is sufficient to trigger major conformational changes and activate RdRp. Chemical screening showed that ribavirin is effective against TSWV, and structural data revealed that ribavirin disrupts both hook-binding and catalytic core formation, locking polymerase in its inactive state. This work provides structural insights into the mechanisms of plant bunyavirus RdRp activation and its dual-targeted site inhibition, facilitating the development of pesticides against plant viruses.
Evolving adeno-associated viruses for gene transfer to the kidney via cross-species cycling of capsid libraries
The difficulty of delivering genes to the kidney has limited the translation of genetic medicines, particularly for the more than 10% of the global population with chronic kidney disease. Here we show that new variants of adeno-associated viruses (AAVs) displaying robust and widespread transduction in the kidneys of mice, pigs and non-human-primates can be obtained by evolving capsid libraries via cross-species cycling in different kidney models. Specifically, the new variants, AAV.k13 and AAV.k20, were enriched from the libraries following sequential intravenous cycling through mouse and pig kidneys, ex vivo cycling in human organoid cultures, and ex vivo machine perfusion in isolated kidneys from rhesus macaques. The two variants transduced murine kidneys following intravenous administration, with selective tropism for proximal tubules, and led to markedly higher transgene expression than parental AAV9 vectors in proximal tubule epithelial cells within human organoid cultures and in autotransplanted pig kidneys. Following ureteral delivery, AAV.k20 efficiently transduced kidneys in pigs and macaques. The AAV.k13 and AAV.k20 variants are promising vectors for therapeutic gene-transfer applications in kidney diseases and transplantation.
SETD1B-mediated broad H3K4me3 controls proper temporal patterns of gene expression critical for spermatid development
Epigenetic programming governs cell fate determination during development through intricately controlling sequential gene activation and repression. Although H3K4me3 is widely recognized as a hallmark of gene activation, its role in modulating transcription output and timing within a continuously developing system remains poorly understood. In this study, we provide a detailed characterization of the epigenomic landscapes in developing male germ cells. We identified thousands of spermatid-specific broad H3K4me3 domains regulated by the SETD1B-RFX2 axis, representing a previously underappreciated form of H3K4me3. These domains, overlapping with H3K27ac-marked enhancers and promoters, play critical roles in orchestrating robust transcription and accurate temporal control of gene expression. Mechanistically, these broad H3K4me3 compete effectively with regular H3K4me3 for transcriptional machinery, thereby ensuring robust levels and precise timing of master gene expression in mouse spermiogenesis. Disruption of this mechanism compromises the accuracy of transcription dosage and timing, ultimately impairing spermiogenesis. Additionally, we unveil remarkable changes in the distribution of heterochromatin marks, including H3K27me3 and H3K9me2, during the mitosis-to-meiosis transition and completion of meiotic recombination, which closely correlates with gene silencing. This work underscores the highly orchestrated epigenetic regulation in spermatogenesis, highlighting the previously unrecognized role of Setd1b in the formation of broad H3K4me3 domains and transcriptional control, and provides an invaluable resource for future studies toward the elucidation of spermatogenesis.
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