Biology subjects

Page, V.

Publications and source records attributed to Page, V..

7 recordsLinked to original sources

bioRxiv2025

Gαq-stimulated gene expression is insensitive to Bromo extra terminal domain inhibitors in HEK 293 cells

Bromodomain and extraterminal domain (BET) family proteins are ubiquitous transcriptional co-activators that function broadly in gene expression programs associated with cellular differentiation, proliferation, and stress responses. Pharmacological inhibition of BET proteins with small molecules that disrupt bromodomain engagement with acetyllysine residues (such as JQ1), or that drive their degradation through the ubiquitin-proteasome system (such as dBET6), ameliorates pathological gene expression in a range of systems and shows promise as a potential therapeutic strategy. BET involvement in gene expression responses is not universal, and understanding of the cell-type and signaling pathway requirements that dictate BET dependence remains incomplete. We previously demonstrated that, in neonatal rat cardiomyocytes, GPCR-induced hypertrophy response depended strongly on the BET protein Brd4 when signaling was coupled to Gs, but not Gq. Here we tested whether Brd4 was differentially responsive to G protein isoforms in HEK 293 cells by expressing Gs or Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Gq induced expression of a group of early response genes and inflammatory genes in a manner largely insensitive to pharmacological BET inhibition, consistent with our previous data in cardiomyocytes. Gs activated a small subset of the Gq-induced genes, but this effect was largely reversed by the BET degrader molecule dBET6. Our data further suggest that there may be general signaling requirements to activate Brd4 across cell types. Highlights- We tested whether Bromodomain and extraterminal domain (BET) family proteins were differentially responsive to G protein isoforms in HEK 293 cells by expressing Gs or Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). - Gq induced expression of a group of genes but was largely insensitive to pharmacological BET inhibition, consistent with our previous data in cardiomyocytes. - Gs activated a small subset of the Gq-induced genes, but was sensitive to the BET degrader molecule dBET6. - Distinct G protein-dependent effects may be conserved across cell types.

molecular biology↗

bioRxiv2023

The Rtf1/Prf1-dependent histone modification axis and Rpb1 C-terminal domain phosphorylation counteract multi-drug resistance in fission yeast

AbstractRNA polymerase II (RNAPII) transcription elongation directs an intricate pattern of histone modifications. Formation of this pattern involves a highly conserved regulatory axis, key components of which are the elongation factor Rtf1, monoubiquitylation of histone H2B (H2Bub1), and methylation of histone H3 on lysine 4 (H3K4me). Direct contact between Rtf1 and the ubiquitylation complex Rad6-Bre1 stimulates H2Bub1, which in turn promotes H3K4me by stimulating the activity of the Set1C/COMPASS methyltransferase complex. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate chromatin and transcription in vivo. We found that mutations affecting multiple components of the Rtf1-H2Bub1-H3K4me axis in the model eukaryote Schizosaccharomyces pombe (fission yeast) caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU). Resistance correlated with a reduced effect of HU on dNTP pools, bypass of the S-phase checkpoint in the presence of HU, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain (CTD) of the RNAPII large subunit Rpb1 led to a similar spectrum of phenotypes. Moreover, all the HU resistant mutants we identified also exhibited resistance to several azole class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 CTD in controlling fungal drug tolerance.

molecular biology↗

bioRxiv2022

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomcyes pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that Spt5 CTD repeat length and phosphorylation have distinct regulatory effects on transcription.

genetics↗

bioRxiv2020

Spt5 phosphorylation and the Rtf1 Plus3 domain promote Rtf1 function through distinct mechanisms

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes co-transcriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9, and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe. We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct, and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps with the pSpt5 binding site and that can interact with single-stranded nucleic acid or with the Polymerase Associated Factor (PAF) Complex in vitro. We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

molecular biology↗

bioRxiv2020

Differential activation of P-TEFb complexes in the development of cardiomyocyte hypertrophy following activation of distinct GPCRs

Pathological cardiac hypertrophy is driven by neurohormonal activation of specific G protein-coupled receptors (GPCRs) in cardiomyocytes and is accompanied by large-scale changes in cardiomyocyte gene expression. These transcriptional changes require activity of positive transcription elongation factor b (P-TEFb), which is recruited to target genes by the bromodomain protein Brd4 or the Super Elongation Complex (SEC). Here we describe GPCR-specific regulation of these P-TEFb complexes and a novel mechanism for activating Brd4 in primary neonatal rat cardiomyocytes. The SEC was required for the hypertrophic response downstream of either the 1-adrenergic receptor (1-AR) or the endothelin receptor (ETR). In contrast, Brd4 inhibition selectively impaired the 1-AR response. This was corroborated by the finding that activation of 1-AR, but not ETR, increased Brd4 occupancy at promoters and super enhancers of hypertrophic genes. Transcriptome analysis demonstrated that activation of both receptors initiated similar gene expression programs, but that Brd4 inhibition attenuated hypertrophic genes more robustly following 1-AR activation. Finally, we show that protein kinase A (PKA) is required for 1-AR stimulation of Brd4 chromatin occupancy. The differential role of the Brd4/P-TEFb complex in response to distinct GPCR pathways has potential clinical implications as therapies targeting this complex are currently being explored for heart failure.

molecular biology↗

bioRxiv2017

Cdk9, Spt5 and histone H2B mono-ubiquitylation cooperate to ensure antisense suppression by the Clr6-CII/Rpd3S HDAC complex

Cyclin-dependent kinase 9 (Cdk9) and histone H2B monoubiquitylation (H2Bub1) are both implicated in elongation by RNA polymerase II (RNAPII). In fission yeast, Cdk9 and H2Bub1 regulate each other through a feedback loop involving phosphorylation of the elongation factor Spt5. Conversely, genetic interactions suggest opposing functions of H2Bub1 and Cdk9 through an Spt5-independent pathway. To understand these interactions, we performed RNA-seq analysis after H2Bub1 loss, Cdk9 inhibition, or both. Either Cdk9 inhibition or H2Bub1 loss increased levels of antisense transcription initiating within coding regions of distinct subsets of genes; ablation of both pathways led to antisense derepression affecting over half the genome. Cdk9 and H2Bub1 cooperate to suppress antisense transcription by promoting function of the Clr6-CII histone deacetylase (HDAC) complex. H2Bub1 plays a second role, in opposition to Clr6-CII, to promote sense transcription in subtelomeric regions. Therefore, functional genomics revealed both collaborative and antagonistic functions of H2Bub1 and Cdk9.

molecular biology↗

bioRxiv2016

Histone gene expression is regulated by histone H2B ubiquitylation in fission yeast

Cell cycle-regulated expression of histone genes is vital for coordination of DNA replication with chromatin assembly. In yeast, histone genes are regulated by transcriptional activation in S phase and transcriptional repression in other phases of the cell cycle, although the mechanisms are poorly understood. Here we describe a role for histone H2B monoubiquitylation (H2Bub1), a histone mark that is linked to RNA polymerase II transcription elongation, in the activation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe. Loss of H2Bub1 is also associated with a delay in S phase progression and reduced expression of the replication initiation factor Cdc18 (ortholog of Cdc6). We provide evidence that H2Bub1 impacts histone gene transcription and acts through a mechanism that involves the 3UTR. Consistent with our previous finding that H2Bub1 is functionally opposed to the elongation factor Cdk9, we also find that the effects of H2Bub1 on histone genes are suppressed by reduction in the activity of Cdk9. Our data suggest that H2Bub1 promotes cell growth by activating cell cycle-regulated genes.

molecular biology↗