Biology subjects

Gamble, M. J.

Publications and source records attributed to Gamble, M. J..

7 recordsLinked to original sources

bioRxiv2025

NMNAT1 Binding at Promoters and Enhancers Couples NAD+ Synthesis to RNA Polymerase II Engagement

Gene expression relies on transcriptional bursts driven by dynamic chromatin-modifying enzymes that depend on metabolites like NAD. Depletion of NAD+ contributes to cancer, metabolic disorders, and aging, emphasizing the significance of tight regulation of NAD+ production in the cell. NMNAT1, a nuclear NAD-synthetase enzyme, supports chromatin-modifying enzymes such as PARP1 and SIRT1; however, its direct role in transcriptional regulation remains unclear. Using integrated multi-omics, we present the first high-resolution, genome-wide study of NMNAT1s regulatory functions. We demonstrate that NMNAT1 binds to the promoters and enhancers of actively transcribed genes involved in DNA replication, cell cycle progression, and chromatin regulation. RNA-seq and CUT&Tag analyses indicate reduced RNA Polymerase II occupancy at downregulated genes in NMNAT1 knockout cells, implicating NMNAT1 in transcriptional activation through Pol II engagement. These findings position NMNAT1 as a key node linking localized NAD production to gene-specific transcription, offering new insights into metabolic regulation of gene expression.

genomics↗

bioRxiv2025

Acetylation of histone H2B on lysine 120 regulates BRD4 binding to intergenic enhancers

BRD4 is a bromodomain-containing transcriptional co-regulator that plays important roles in driving transcription by binding to histone acetyl-lysines at enhancers and promoters while recruiting additional transcriptional cofactors. While the mechanisms by which BRD4 regulates transcription have been explored, the critical acetylations primarily responsible for targeting it to chromatin remain unclear. Through a machine learning approach, we determined that distinct sets of histone acetylations dominate the prediction of chromatin accessibility and BRD4 binding in distinct chromatin contexts (e.g. intergenic enhancers, gene body enhancers and promoters). Using human fibroblasts engineered to predominantly express specific histones with lysine-to-arginine mutations, we demonstrate that one such acetylation, H2BK120ac, is required to recruit BRD4 specifically to intergenic enhancers, while not affecting chromatin accessibility. Loss of H2BK120ac did not affect BRD4 binding to either promoters or gene body enhancers, demonstrating that the rules governing BRD4 recruitment to regulatory regions depends on the specific genomic context. Highlighting the importance of H2BK120ac in directing BRD4 recruitment, we found that expression of the H2BK120R mutant significantly reduces the phenotypes driven by BRD4-NUT, an oncogenic fusion protein that drives NUT midline carcinoma. This work demonstrates the critical nature that genomic context plays in BRD4 recruitment to distinct classes of regulatory elements, and suggests that intergenic and gene body enhancers represent classes of functional distinct elements.

molecular biology↗

bioRxiv2024

Platelet PI3Kβ regulates breast cancer metastasis

Platelets promote tumor metastasis by several mechanisms. Platelet-tumor cell interactions induce the release of platelet cytokines, chemokines, and other factors that promote tumor cell epithelial-mesenchymal transition and invasion, granulocyte recruitment to circulating tumor cells (CTCs), and adhesion of CTCs to the endothelium, assisting in their extravasation at metastatic sites. Previous studies have shown that platelet activation in the context of thrombus formation requires the Class IA PI 3-kinase PI3K{beta}. We now define a role for platelet PI3K{beta} in breast cancer metastasis. Platelet PI3K{beta} is essential for platelet-stimulated tumor cell invasion through Matrigel. Consistent with this finding, in vitro platelet-tumor cell binding and tumor cell-stimulated platelet activation are reduced in platelets isolated from PI3K{beta} mutant mice. RNAseq and proteomic analysis of human breast epithelial cells co-cultured with platelets revealed that platelet PI3K{beta} regulates the expression of EMT and metastasis-associated genes in these cells. The EMT and metastasis-associated proteins PAI-1 and IL-8 were specifically downregulated in co-cultures with PI3K{beta} mutant platelets. PI3K{beta} mutant platelets are impaired in their ability to stimulate YAP and Smad2 signaling in tumor cells, two pathways regulating PAI-1 expression. Finally, we show that mice expressing mutant PI3K{beta} show reduced spontaneous metastasis, and platelets isolated from these mice are less able to stimulate experimental metastasis in WT mice. Taken together, these data support a role for platelet PI3K{beta} in promoting breast cancer metastasis and highlight platelet PI3K{beta} as a potential therapeutic target. SignificanceWe demonstrate that platelet PI3K{beta} regulates metastasis, broadening the potential use of PI3K{beta}-selective inhibitors as novel agents to treat metastasis.

cancer biology↗

bioRxiv2024

Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB

Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) --but not type I PRMT inhibition--leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.

molecular biology↗

bioRxiv2021

Type I PRMTs and PRMT5 Inversely Regulate Post-Transcriptional Intron Detention

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed SKaTER-seq method, analysis of co-transcriptional splicing revealed that PRMT inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternative splicing of polyadenylated RNA--particularly intron retention (RI). Investigation of RI following inhibition of nascent transcription demonstrated that PRMTs inversely regulate RI post-transcriptionally. Subsequent proteomic analysis of chromatin-associated polyadenylated RNA identified aberrant binding of the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition. Conversely, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Closer examination of subcellular fractions indicated that RI were isolated to the nucleoplasm and chromatin. Together, these data demonstrate that PRMTs regulate the post-transcriptional processing of nuclear, detained introns through Sm and CHTOP arginine methylation.

cancer biology↗

bioRxiv2021

Spatial organization of transcript elongation and splicing kinetics

The organization of the genome in three-dimensional space has been shown to play an important role in gene expression. Specifically, facets of genomic interaction such as topologically associated domains (TADs) have been shown to regulate transcription by bringing regulatory elements into close proximity1. mRNA production is an intricate process with multiple control points including regulation of Pol II elongation and the removal of non-coding sequences via pre-mRNA splicing2. The connection between genomic compartments and the kinetics of RNA biogenesis and processing has been largely unexplored. Here, we measure Pol II elongation and splicing kinetics genome-wide using a novel technique that couples nascent RNA-seq with a mathematical model of transcription and co-transcriptional RNA processing. We uncovered multiple layers of spatial organization of these rates: the rate of splicing is coordinated across introns within individual genes, and both elongation and splicing rates are coordinated within TADs, as are alternative splicing outcomes. Overall, our work establishes that the kinetics of transcription and splicing are coordinated by the spatial organization of the genome and suggests that TADs are a major platform for coordination of alternative splicing.

molecular biology↗

bioRxiv2020

Type I PRMTs and PRMT5 Independently Regulate Both snRNP Arginine Methylation and Post-Transcriptional Splicing

Protein arginine methyltransferases (PRMTs) methylate histones, splicing factors, and many other nuclear proteins. Type I enzymes (PRMT1-4,6,8) catalyze mono- (Rme1/MMA) and asymmetric (Rme2a/ADMA) dimethylation; Type II enzymes (PRMT5,9) catalyze mono- and symmetric (Rme2s/SDMA) dimethylation. Misregulation of PRMTs in multiple types of cancers is associated with aberrant gene expression and RNA splicing. To understand the specific mechanisms of PRMT activity in splicing regulation, we treated cells with the PRMT5 inhibitor GSK591 and the Type I inhibitor MS023 and probed their transcriptomic consequences. We discovered that Type I PRMTs and PRMT5 inversely regulate core spliceosomal Sm protein Rme2s and intron retention. Loss of Sm Rme2s is associated with the accumulation of polyadenylated RNA containing retained introns and snRNPs on chromatin. Conversely, increased Sm Rme2s correlates with decreased intron retention and chromatin-association of intron-containing polyadenylated RNA. Using the newly developed SKaTER-seq model, comprehensive and quantitative analysis of co-transcriptional splicing revealed that either Type I PRMT or PRMT5 inhibition resulted in slower splicing rates. Surprisingly, altered co-transcriptional splicing kinetics correlated poorly with ultimate changes in alternatively spliced mRNA. Quantitation of retained intron decay following inhibition of nascent transcription revealed that Type I PRMTs and PRMT5 reciprocally regulate post-transcriptional splicing efficiency.

cell biology↗