Transcription activation depends on the length of the RNA polymerase II C‐terminal domain

Eukaryotic RNA polymerase II (Pol II) contains a tail‐like, intrinsically disordered carboxy‐terminal domain (CTD) comprised of heptad‐repeats, that functions in coordination of the transcription cycle and in coupling transcription to co‐transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre‐mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter‐proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer‐dependent recruitment of Pol II to target genes for their rapid activation

Nuclear target search at the single molecule level: protein interactions define the exploration landscape

Gene regulation relies on highly mobile transcription factors (TFs) exploring the nucleoplasm in search of their targets. Our view of the nucleus has evolved from that of an isotropic and homogenous reactor to that of a highly organized yet very dynamic organelle. However important questions remain on how these regulatory factors explore the nuclear environment in search of their DNA or protein targets, and how their exploration strategy affects the kinetics of transcriptional regulation. We implemented a single-molecule tracking assay to determine the TFs dynamics using photoactivatable tags in human cells. We investigated the mobility of several nuclear proteins, including the transcription factor c-Myc and the elongation factor P-TEFb. We found that, while their diffusion speed was comparable, these proteins largely differed in terms of their exploration geometry. We discovered that c-Myc is a global explorer diffusing in the nucleus without spatial constraints. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment, constrained by a fractal nuclear architecture. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. We also measured the mobility of a P-TEFb mutant in which the interaction with the CTD of the RNA Pol II was truncated. In this case, the single-molecule experiments suggested a global exploration of the P-TEFb mutant, consistent with free diffusion. Our observations are in line with a model in which the exploration geometry of TFs is constrained by their interactions and not by exclusion properties. Our findings have strong implications on how proteins react in the nucleus and how their function can be regulated in space and time

Promoter methylation and large intragenic rearrangements of DPYD are not implicated in severe toxicity to 5-fluorouracil-based chemotherapy in gastrointestinal cancer patients

<p>Abstract</p> <p>Background</p> <p>Severe toxicity to 5-fluorouracil (5-FU) based chemotherapy in gastrointestinal cancer has been associated with constitutional genetic alterations of the dihydropyrimidine dehydrogenase gene (<it>DPYD</it>).</p> <p>Methods</p> <p>In this study, we evaluated <it>DPYD </it>promoter methylation through quantitative methylation-specific PCR and screened <it>DPYD </it>for large intragenic rearrangements in peripheral blood from 45 patients with gastrointestinal cancers who developed severe 5-FU toxicity. <it>DPYD </it>promoter methylation was also assessed in tumor tissue from 29 patients</p> <p>Results</p> <p>Two cases with the IVS14+1G > A exon 14 skipping mutation (c.1905+1G > A), and one case carrying the 1845 G > T missense mutation (c.1845G > T) in the DPYD gene were identified. However, <it>DPYD </it>promoter methylation and large <it>DPYD </it>intragenic rearrangements were absent in all cases analyzed.</p> <p>Conclusions</p> <p>Our results indicate that <it>DPYD </it>promoter methylation and large intragenic rearrangements do not contribute significantly to the development of 5-FU severe toxicity in gastrointestinal cancer patients, supporting the need for additional studies on the mechanisms underlying genetic susceptibility to severe 5-FU toxicity.</p

Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs

Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs

RNA polymerase II clustering through carboxy-terminal domain phase separation.

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation

Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon–exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS

Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19