Biology subjects

Wilson, T. E.

Publications and source records attributed to Wilson, T. E..

11 recordsLinked to original sources

bioRxiv2026

Characterizing nascent readthrough transcripts in human cells

Termination of transcription by RNA polymerase II (RNAPII) is critical to appropriately define transcriptome and proteome identity; yet mechanisms of termination regulation are incompletely understood. Current models suggest critical roles for the exonuclease XRN2, which is posited to "torpedo" out RNAPII after degrading uncapped and RNAPII-tethered nascent transcripts and posit an allosteric model wherein elongating RNAPII loses its processivity downstream of the transcription end site (TES) via unknown mechanisms. Here, we used nascent RNA Bru-seq to interrogate transcription termination across 16 human cell lines of distinct lineages, as part of the ENCODE 4 project, to identify key regulatory modes. We find that > 75% of protein-coding genes engage in readthrough transcription under homeostatic conditions. Exposing human cells to oxidative stress or hyperosmotic conditions or splicing inhibitors cause an increase in readthrough transcripts globally. Using an auxin-inducible degradation approach we find that depletion of BRD4, MED14 or XRN2 increases the extent and pervasiveness of readthrough transcripts while depletion of SUPT16H results in shorter readthrough transcripts. Our study provides a comprehensive view of homeostatic readthrough transcripts in human cells and delineates key components regulating transcription termination.

molecular biology↗

bioRxiv2026

High-fidelity rare structural variant detection with HiFiRE3 reduced representation via restriction enzyme ends

High-fidelity detection of rare structural variants (SVs) remains challenging because library preparation and sequencing techniques generate artifactual junctions that obscure true single-molecule events. Here, we present HiFiRe3, an error-minimized sequencing framework that combines artifact-aware library design with error suppression and correction strategies to enable rare SV detection and frequency assessment across long and short-read sequencing platforms. We first systematically characterized major classes of SV artifacts, including chimeric PCR products, intermolecular ligation, sequencing platform-specific artifacts, and mapping errors. HiFiRe3 supports error correction of these artifact junctions by combining reduced representation restriction fragments with pre-ligation size selection to enable computational filtering via independent forced restriction enzyme end (FREE) and <1N size logics. In nanopore libraries, these approaches enabled targeted detection of single-molecule SVs at replication stress hotspots in cultured human cells exposed to genotoxicants and in long genes in untreated mouse brains, while markedly reducing singleton translocation artifacts. HiFiRe3 extends to PacBio sequencing for joint SV and SNV error correction and to short-read platforms for cost-efficient high-fidelity nonhomologous SV analysis. Together, HiFiRe3 is a flexible framework for accurately detecting rare genomic structural variation with broad applicability to targeted and genome-wide studies by selective application of its error correction approaches.

genomics↗

bioRxiv2026

3D chromatin compartment of round spermatids encodes the spatiotemporal program of histone-to-protamine exchange in spermiogenesis

Sperm formation requires a radical chromatin reorganization, where nucleosomes are replaced by transition proteins (TNPs) and subsequently by protamines (PRM1 and PRM2). Although essential for fertility, the regulatory logic governing this exchange is unknown, but its presumed to be stochastic and unregulated. Using endogenously tagged PRM mouse models and stage-resolved, genome-wide profiling, we revise the order of histone-to-protamine exchange and show that the chromatin remodeling process is highly programmed. Imaging and biochemical experiments reveal a direct histone-to-PRM1 exchange, while TNPs appear after PRM1 but precede PRM2 incorporation. This temporal uncoupling of PRM1 and PRM2 incorporation coincides with dynamic, region-specific chromatin remodeling that is not governed by histone acetylation but is instructed by the three-dimensional nuclear architecture of round spermatids. Therefore, by integrating ATAC-seq, CUT&Tag, and Hi-C, we define a compartment-encoded "blueprint" that prescribes the assembly of the mature sperm epigenome and establishes a complex molecular hierarchy for the histone-to-protamine exchange.

genomics↗

bioRxiv2026

MRE11 suppresses germline mutagenesis at meiotic double-strand breaks in mice

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as [~]21 bp. The tyrosyl DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

molecular biology↗

bioRxiv2024

Isoform and pathway-specific post-transcriptional RNA processing in human cells

Steady-state levels of RNA transcripts are controlled by their rates of synthesis and degradation. Here we used nascent RNA Bru-seq and BruChase-seq to profile RNA dynamics across 16 human cell lines as part of ENCODE4 Deeply Profiled Cell Lines collection. We show that RNA turnover dynamics differ widely between transcripts of different genes and between different classes of RNA. Gene set enrichment analysis (GSEA) revealed that transcripts encoding proteins belonging to the same pathway often show similar turnover dynamics. Furthermore, transcript isoforms show distinct dynamics suggesting that RNA turnover is important in regulating mRNA isoform choice. Finally, splicing across newly made transcripts appears to be cooperative with either all or none type splicing. These data sets generated as part of ENCODE4 illustrate the intricate and coordinated regulation of RNA dynamics in controlling gene expression to allow for the precise coordination of cellular functions.

molecular biology↗

bioRxiv2024

POLQ mediates replication-stress induced structural variant formation throughout common fragile sites during mitosis

Genomic structural variants (SVs) greatly impact human health and disease, but much is unknown about their generative mechanisms, especially for the large class of nonrecurrent alterations. Common fragile sites (CFSs) are unstable loci that provide a model for SV formation, especially large deletions, under replication stress. We studied SV junction formation as it occurred in cells by applying error-minimized capture sequencing to CFS DNA harvested during replication stress. SV junctions formed throughout CFS genes at a 5-fold higher rate after cells passed from G2 into M-phase. Neither SV formation nor CFS expression depended on mitotic DNA synthesis (MiDAS), an error-prone form of conservative replication active at CFSs. Instead, analysis of tens of thousands of de novo SV junctions combined with DNA repair pathway inhibition revealed a primary role for DNA polymerase theta (POLQ)-mediated end-joining (TMEJ) in M-phase SV formation. We propose an important role for TMEJ in nonrecurrent SV formation genome wide.

genetics↗

bioRxiv2024

Characterizing nascent transcription patterns of PROMPTs, eRNAs, and readthrough transcripts in the ENCODE4 deeply profiled cell lines

Arising as co-products of canonical gene expression, transcription-associated lincRNAs, such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and readthrough (RT) transcripts, are often regarded as byproducts of transcription, although they may be important for the expression of nearby genes. We identified regions of nascent expression of these lincRNA in 16 human cell lines using Bru-seq techniques, and found distinctly regulated patterns of PROMPT, eRNA, and RT transcription using the diverse biochemical approaches in the ENCODE4 deeply profiled cell lines collection. Transcription of these lincRNAs was influenced by sequence-specific features and the local or 3D chromatin landscape. However, these sequence and chromatin features do not describe the full spectrum of lincRNA expression variability we identify, highlighting the complexity of their regulation. This may suggest that transcription-associated lincRNAs are not merely byproducts, but rather that the transcript itself, or the act of its transcription, is important for genomic function.

molecular biology↗

bioRxiv2022

svCapture: Efficient and specific detection of very low frequency structural variant junctions by error-minimized capture sequencing

Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to strategies for comparable detection of rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from cell samples with known SV properties, we demonstrate that Duplex Sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of even single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and the high base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of sub-clonal and newly created SVs, suggesting end joining as a predominant formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.

genomics↗

bioRxiv2021

Mapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

Resection of the 5-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing in which molecular identifiers allow exact counting of cleaved 5 ends at base pair resolution. Mutant strains, including exo1{Delta}, mre11-H125N, exo1{Delta} and exo1{Delta} sgs1{Delta}, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked a possible motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1{Delta} sgs1{Delta} yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

genetics↗

bioRxiv2021

Multiple metabolic signals including AMPK and PKA regulate glucose-stimulated double strand break resection in yeast

DNA double strand breaks (DSBs) are cytotoxic lesions repaired by non-homologous end joining (NHEJ) and homologous recombination (HR), with 5 strand resection being the committed step in transition from NHEJ to HR. We previously discovered that gal1 yeast, which cannot metabolize galactose, were unable to perform efficient 5 resection even though DSBs were formed. Adding glucose or restoring GAL1 restored resection, suggesting that carbon source metabolism signals to DSB repair. Here we demonstrate that any fermentable carbon source, including raffinose, can stimulate resection and that the stimulatory effect of glucose was associated with decreased, not increased, cellular ATP. The effect was cell cycle dependent and did not occur in G1, while glucose augmented the G2/M checkpoint arrest even in cells deficient in resection. AMP-activated protein kinase pathway mutants showed only low basal resection despite glucose addition but had normal checkpoint arrest, indicating a primary role for Snf1 specifically in glucose-stimulated resection. The metabolic inputs to resection were multifactorial, however, with loss of the transcriptional repressor Mig1 leading to increased basal resection, three distinct patterns of deficiency with loss of the protein kinase A catalytic subunits, Tpk1, Tpk2 andTpk3, and a resection delay in yeast lacking the lysine demethylase Rph1 that helped separate early and late phase responses to glucose. These results reveal multiple interrelated metabolic signals that optimize DSB resection efficiency while independently amplifying the G2/M checkpoint response.

genetics↗

bioRxiv2020

Role of bradykinin type 2 receptors in human sweat secretion: translational evidence does not support a functional relationship

Bradykinin increases skin blood flow via a cGMP mechanism but its role in sweating in vivo is unclear. There is a current need to translate cell culture and non-human paw pad studies into in vivo human preparations to test for therapeutic viability for disorders affecting sweat glands. Protocol 1: physiological sweating was induced in 10 healthy subjects via perfusing warm (46-48{degrees}C) water through a tube-lined suit while bradykinin type 2 receptor (B2R) antagonist (HOE-140; 40 M) and only the vehicle (lactated Ringers) were perfused intradermally via microdialysis. Heat stress increased sweat rate (HOE-140 = +0.79{+/-}0.12 and vehicle = +0.64{+/-}0.10 mg/cm2/min), but no differences were noted with B2R antagonism. Protocol 2: pharmacological sweating was induced in 6 healthy subjects via intradermally perfusing pilocarpine (1.67 mg/ml) followed by the same B2R antagonist approach. Pilocarpine increased sweating (HOE-140 = +0.38{+/-}0.16 and vehicle = +0.32{+/-}0.12 mg/cm2/min); again no differences were observed with B2R antagonism. Lastly, 5 additional subjects were recruited for various control experiments which identified that a functional dose of HOE-140 was utilized and it was not sudorific during normothermic conditions. These data indicate B2R antagonists do not modulate physiologically-or pharmacologically-induced eccrine secretion volumes. Thus, B2R agonist/antagonist development as a potential therapeutic target for hypo- and hyperhidrosis appears unwarranted.

physiology↗