Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated in diseases, including Sotos syndrome. Despite the impacts of H3K36me2 on H3K27me3 and DNA methylation, the direct role of NSD1 in transcriptional regulation remains largely unknown. Here, we show that NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1 enhancer association is conferred by a tandem quadruple PHD (qPHD)-PWWP module, which recognizes p300-catalyzed H3K18ac. By combining acute NSD1 depletion with time-resolved epigenomic and nascent transcriptomic analyses, we demonstrate that NSD1 promotes enhancer-dependent gene transcription by facilitating RNA polymerase II (RNA Pol II) pause release. Notably, NSD1 can act as a transcriptional coactivator independent of its catalytic activity. Moreover, NSD1 enables the activation of developmental transcriptional programs associated with Sotos syndrome pathophysiology and controls embryonic stem cell (ESC) multilineage differentiation. Collectively, we have identified NSD1 as an enhancer-acting transcriptional coactivator that contributes to cell fate transition and Sotos syndrome development.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.
Copyright © 2021 Elsevier Inc. All rights reserved.

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.
Copyright © 2020 Elsevier Inc. All rights reserved.

RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.
Copyright © 2020 Elsevier Inc. All rights reserved.