Biology subjects

Gillespie, M. N.

Publications and source records attributed to Gillespie, M. N..

3 recordsLinked to original sources

bioRxiv2025

Analysis of alternative splicing uncovers a vastly expanded transcriptomic response to hypoxia in human vascular endothelial cells

Hypoxia is a fundamental pathophysiological stimulus that plays important roles in multiple cardiopulmonary diseases. During hypoxic stress, cells adapt by undergoing widespread transcriptional reprogramming. The conventional approach to investigating this response has largely involved RNA-seq analysis which typically quantifies transcriptional output at the level of individual genes. However, as most mammalian genes encode multiple transcripts that are divergently regulated by alternative splicing, consolidating these discrete features to the gene level can obscure critical changes within the transcriptome that are largely unappreciated. To more accurately define the role of individual transcript usage during hypoxia, herein we employed three different analytical strategies that collectively identified thousands of instances of alternative splicing in human endothelial cells undergoing hypoxic stress. Notably, the set of differentially utilized transcripts displayed minimal overlap with the genes identified to be differentially expressed by conventional RNA-seq analysis, indicating divergent usage of individual transcripts does not reliably culminate in a detectable change in overall expression. This outcome is particularly clear at the earliest time-point of hypoxia where we found the transcriptional response was mediated almost exclusively by alternative splicing. A subset of these acutely responsive genes was variably detected as differentially expressed or alternatively spliced at later time points, demonstrating hypoxic transcription is highly dynamic and temporally complex. Further, the number of genes in pathways incriminated in the hypoxic response was also expanded considerably when alternatively spliced transcripts were included in the analysis, suggesting distinct attributes of the hypoxic transcriptome are unveiled by transcript level assays that would otherwise be obfuscated by standard RNA-seq analyses that summarize expression to the gene level. Collectively, these results strongly point to alternative splicing being a significant, albeit understudied, component of the transcriptional response to hypoxic stress which is vastly more complicated than previously thought.

genomics↗

bioRxiv2025

The Cistrome Response to Hypoxia in Human Umbilical Vein Endothelial Cells

Hypoxic stress triggers transcriptional signaling mainly through hypoxia-inducible transcription factors (HIFs), which bind hypoxia response elements (HREs) in gene regulatory regions. However, only a small proportion ([~]1%) of known HREs are occupied by HIFs during hypoxia, suggesting the involvement of additional hypoxia-responsive factors. To address this gap, we utilized MNase-defined cistrome Occupancy Analysis sequencing (MOA-seq), with the term cistrome referring to all genomic regions where transcription factors and other trans-acting regulators are bound to cis-acting elements across the genome for a particular cell type or treatment. This MNase-based assay enables genome-wide, high-resolution (<30 bp) identification of transcription factor (TF) occupancy footprints embedded within larger regions, most of which were previously annotated as open or accessible chromatin. Applying this in situ cistrome mapping to fixed nuclei from endothelial cells under normoxia or hypoxia (1, 3, or 24 hours) revealed thousands of hypoxia-responsive genomic sites with dynamic TF footprints. The affected genes were enriched in canonical hypoxia-induced pathways, such as angiogenesis. Motif analysis identified over 100 candidate TFs potentially mediating these multifaceted genomic responses. By grouping hypoxia-modified occupancy signals across the hypoxia exposure times, we clustered differentially occupied MOA sites into defined 10 distinct TF kinetic clusters, half of which were associated with HIF1A. HIF1A-proximal binding sites suggested co-activators, while non-HIF1A clusters pointed to additional TFs that may have HIF1A-independent roles. This analysis provides insight into how multiple TF networks coordinate hypoxia responses and highlights the power of cistrome profiling to deepen our understanding of the complex genomic response to low oxygen conditions. KEY POINTSO_LIMOA-seq mapped TF occupancy at 21,765 sites in normoxia, including 7,444 beyond the known ENCODE cCREs. C_LIO_LIHypoxia for 1, 3, and 24h changes the cistrome occupancy at thousands of genes. C_LIO_LIClustering analysis of hypoxia-responsive footprints consolidated cistrome kinetics into HIF1A-associated and HIF1A-independent TFs. C_LI

genomics↗

bioRxiv2024

Long G4-rich enhancer physically interacts with EXOC3 promoter via a G4:G4 DNA-based mechanism

Enhancers are genomic sequences that function as regulatory elements capable of increasing the transcription of a given gene often located at a considerable distance. The broadly accepted model of enhancer activation involves bringing an enhancer-bound activator protein complex into close spatial proximity to its target promoter through chromatin looping. Equally relevant to the work described herein, roles for guanine (G) rich sequences in transcriptional regulation are now widely accepted. Non-coding G-rich sequences are commonly found in gene promoters and enhancers, and various studies have described specific instances where G-rich sequences regulate gene expression via their capacity to form G-quadruplex (G4) structures under physiological conditions. In light of this, our group previously performed a search for long human genomic stretches significantly enriched for minimal G4 motifs (referred to as LG4s herein) leading to the identification of 301 LG4 loci with a density of at least 80 GGG repeats / 1,000 basepairs (bp) and averaging 1,843 bp in length. Further, in agreement with previous reports indicating that minimal G4s are highly enriched in promoters and enhancers, we found 217/301 LG4 sequences overlap a GeneHancer annotated enhancer, and the gene promoters regulated by these LG4 enhancers were found to be similarly, markedly enriched with G4-capable sequences. Importantly, while the generally accepted model for enhancer:promoter specificity maintains that interactions are dictated by enhancer- and promoter-bound transcriptional activator proteins, the current study was designed to test an alternative hypothesis: that LG4 enhancers physically interact with their cognate promoters via a direct G4:G4 DNA-based mechanism. As such, this work employs a combination of informatic mining and locus-specific immunoprecipitation strategies to establish the spatial proximity of enhancer:promoter pairs within the nucleus then biochemically confirms the ability of individual LG4 ssDNAs to directly and specifically interact with DNA sequences found in their target promoters. In addition, we also identify four single nucleotide polymorphisms (SNPs), occurring within a LG4 enhancer on human chromosome 5, significantly associated with Cystic Fibrosis (CF) lung disease severity (avg. p value = 2.83E-9), presumably due to their effects on the expressions of CF-relevant genes directly regulated by this LG4 enhancer (e.g., EXOC3 and CEP72). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/577212v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1fee785org.highwire.dtl.DTLVardef@11f0a9org.highwire.dtl.DTLVardef@1cf198corg.highwire.dtl.DTLVardef@1963a55_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief: LG4 enhancers physically interact with gene promoters by forming composite G4 structures where both the LG4 and cognate promoter contribute half of the necessary sequence for G4 formation.

genetics↗