Biology subjects

Rowley, M. J.

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

11 recordsLinked to original sources

bioRxiv2026

Guanine content in the CTCF motif encodes a vulnerability to persistent, alcohol-induced redistribution

Alcohol consumption is a leading contributor to the global disease burden, yet its molecular consequences remain incompletely understood. Ethanols first metabolite, acetaldehyde, modifies guanine to form DNA adducts and DNA-protein crosslinks that block elongating RNA polymerase II. Because acetaldehyde acts specifically on guanine, we hypothesized that chromatin features would be disrupted in a sequence-dependent rather than stochastic manner. Here we confirm that alcohol exposure displaces elongating RNA polymerase II (RNAPIIs2p) from gene bodies, relocating to promoters. In addition to this confirmation, we found that RNAPIIs2p also relocates to discrete guanine-rich intergenic CTCF sites. Loci with high guanine content at the CTCF motifs zinc finger 5 (ZF5) binding position showed the largest increase in both CTCF and cohesin (RAD21), with CTCF occupancy changes persisting even six days after alcohol withdrawal. Surprisingly, using Hi-C, we found that these changes to CTCF and cohesin do not impact the pronounced structural CTCF loops, but are instead primarily associated with altered guanine-rich enhancer-promoter contacts that connect to lasting changes to gene expression. Beyond our cell-line model, the same sequence rule accounts for altered CTCF occupancy in a mouse starvation (metabolic stress) model and in the placentae after paternal alcohol exposure. Together these findings link alcohol exposure to sequence-directed changes to chromatin whose rules are conserved in alcohol-associated disease.

molecular biology↗

bioRxiv2025

The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

Runt-related Transcription Factor 1 (RUNX1) is essential for definitive hematopoiesis and is among the most frequently mutated genes in leukemia. Previous work from our lab demonstrated that Histone Deacetylase 1 (HDAC1), a known RUNX1 partner, is unexpectedly required for active transcription suggesting a non-histone role for HDAC1 regulating components of the RUNX1 complex. Here, we use proteomics, genomics, and long-read transcriptomics to identify novel RUNX1 interacting partners and decipher their role in gene regulation and RNA splicing in leukemia cells. We demonstrate that Polypyrimidine Tract Binding Protein 1 (PTBP1) interacts with RUNX1 in an HDAC1 dependent manner. Chromatin profiling revealed extensive genome-wide overlap in sites occupied by RUNX1 and PTBP1, with significant enrichment at promoters of actively transcribed genes. Loss of PTBP1 in AML cells led to widespread alterations in RNA splicing and decreased expression of genes whose promoters are bound by both factors, including metabolic genes. In agreement with these findings, we found that loss of PTBP1 reduced glycolysis and glucose uptake and ultimately caused cell death. Based on our data, we propose that the interaction between RUNX1 and PTBP1 facilitates expression of metabolic proteins essential for leukemia cell growth and survival. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/654547v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@74e3b9org.highwire.dtl.DTLVardef@12a090org.highwire.dtl.DTLVardef@eb5a48org.highwire.dtl.DTLVardef@17465dc_HPS_FORMAT_FIGEXP M_FIG C_FIG KEY POINTSO_LIPTBP1 binds RUNX1 in a HDAC1-dependent manner and co-localizes to the promoters of target genes in leukemia cells. C_LIO_LILoss of PTBP1 decreases expression of key metabolic genes, resulting in decreased cell growth and glycolysis, increased sensitivity to chemotherapy, and cell death. C_LI

cancer biology↗

bioRxiv2025

ECD, a novel androgen receptor target promotes prostate cancer tumorigenesis by regulating glycolysis

Androgen receptor (AR)-mediated signaling is essential for PC tumorigenesis. In the TCGA database we observed a positive correlation between ECD and AR expression. Consistently, Dihydrotestosterone (DHT) treatment of PC cell lines increased ECD mRNA and protein levels, and AR knockdown (KD) reduced ECD expression. Bioinformatic analysis predicted three consensus androgen response elements in the ECD promoter, and DHT treatment increased AR occupancy at the ECD promoter, and enhanced ECD promoter activity. Enzalutamide treatment decreased ECD levels, and ECD knockout (KO) in PC cells reduced oncogenic traits, suggesting a functional role of ECD to maintain PC oncogenesis. ECD mRNA and protein are overexpressed in PC patient tissues, and its overexpression predicts shorter survival. Overexpression of ECD in PC cell lines enhanced the oncogenic traits in vitro and developed faster and larger highly proliferative xenograft tumors. RNA-seq analysis of mouse tumors revealed an increase in mRNA levels of several glycolytic genes. ECD associates with mRNA of key glycolytic genes and is required for their stability, consistent with our recent demonstration of ECD is an RNA binding protein. Higher glucose uptake and glycolysis was seen upon ECD overexpression in PC cells. Together, we demonstrate the role of a novel AR target gene ECD in PC tumorigenesis.

cancer biology↗

bioRxiv2025

ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

The ecdysoneless (ECD) mRNA and protein are overexpressed in breast cancer (BC), and its overexpression correlates with poor prognosis and short patient survival, particularly in ERBB2/HER2-positive BC. This study investigates the co-operative oncogenic mechanism of ECD and ERBB2 by deriving transgenic mice overexpressing ECD and/or ERBB2 (huHER2) in mammary epithelium under MMTV promoter, as well as human mammary immortal epithelial cell lines (hMECs) overexpressing ECD and/or ERBB2. While huHER2Tg mice developed more homogenous solid nodular carcinomas, double transgenic mice (ECD;huHER2Tg) developed heterogenous and histologically aggressive mammary tumors with basal-like phenotype and epithelial mesenchymal transition (EMT) features, like ECDTg tumors, resembling more to patient tumors. Importantly, transcriptomic profile of ECD;huHER2Tg tumors revealed upregulation of two major oncogenic pathways, unfolded protein response (UPR) and glycolysis. Similarly, hMECs expressing both ECD and ERBB2 as compared to single gene expressing cells showed increase in oncogenic traits, and RNA-seq analysis showed a significant upregulation of glycolysis and UPR pathways. ECD is an RNA binding protein, and directly associates with three key glycolytic enzymes (LDHA, PKM2 and HK2) and mRNA of a major UPR regulated gene GRP78, that results in increased mRNA stability. Lastly, we show an increase in glucose uptake and enhanced glycolytic rate in ECD+ERBB2-overexpressing cells as compared to ECD- or ERBB2-overexpressing hMECs. Taken together, our findings support a co-operative role of ECD and ERBB2 in oncogenesis by enhancing two major oncogenic pathways, UPR and glycolysis. SignificanceThis study provides mechanistic insights that overexpression of ECD in ERBB2+ breast cancer patients correlates with shorter patient survival, by identifying direct ECD binding to mRNAs for UPR and glycolysis pathways.

cancer biology↗

bioRxiv2025

ECD functions as a novel RNA-binding protein to regulate mRNA splicing

Human ecdysoneless protein (ECD) plays an essential role in regulating cell cycle progression and cell survival. ECD has previously been implicated in RNA splicing through its association with spliceosomal proteins. Here, using EMSA, fluorescence polarization assays, and mutational analysis, we demonstrate that ECD directly binds to RNA. Enhanced CLIP-seq analysis identified a broad repertoire of mRNAs bound to ECD in cells. RNA-seq analyses revealed that ECD depletion leads to widespread splicing aberrations and altered gene expression. ECD binding to RNAs was enriched near splice sites, and a substantial fraction of ECD-bound transcripts exhibited splicing defects upon ECD depletion. ECD associates with and stabilizes the U5 snRNP complex specific proteins. While depletion of ECD reduced the levels of key U5-specifc proteins, these proteins exhibited an increased association with the R2TP complex in knockout cells. Notably, we found ECD to directly bind to U5 snRNA, and an RNA binding defective mutant of ECD ({Delta}135-148) failed to rescue the reduced levels of U5-specific proteins or the proliferation defect induced by ECD depletion. Collectively, these findings demonstrate that ECD binds to RNAs, including the U5 snRNA, and that RNA-binding is required for ECD to stabilize the U5 snRNP and for cellular functions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/634785v4_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@f5aa4forg.highwire.dtl.DTLVardef@80606borg.highwire.dtl.DTLVardef@3a4c8eorg.highwire.dtl.DTLVardef@1780b0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

HiCrayon reveals distinct layers of multi-state 3D chromatin organization

The co-visualization of chromatin conformation with 1D omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayons visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization. Local version: https://github.com/JRowleyLab/HiCrayon Web version: https://jrowleylab.com/HiCrayon

genomics↗

bioRxiv2023

Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample

Ancient DNA (aDNA) sequencing analysis typically involves alignment to a modern reference genome assembly from a related species. Since aDNA molecules are fragmentary, these alignments yield information about small-scale differences, but provide no information about larger features such as the chromosome structure of ancient species. We report the genome assembly of a female Late Pleistocene woolly mammoth (Mammuthus primigenius) with twenty-eight chromosome-length scaffolds, generated using mammoth skin preserved in permafrost for roughly 52,000 years. We began by creating a modified Hi-C protocol, dubbed PaleoHi-C, optimized for ancient samples, and using it to map chromatin contacts in a woolly mammoth. Next, we developed "reference-assisted 3D genome assembly," which begins with a reference genome assembly from a related species, and uses Hi-C and DNA-Seq data from a target species to split, order, orient, and correct sequences on the basis of their 3D proximity, yielding accurate chromosome-length scaffolds for the target species. By means of this reference-assisted 3D genome assembly, PaleoHi-C data reveals the 3D architecture of a woolly mammoth genome, including chromosome territories, compartments, domains, and loops. The active (A) and inactive (B) genome compartments in mammoth skin more closely resemble those observed in Asian elephant skin than the compartmentalization patterns seen in other Asian elephant tissues. Differences in compartmentalization between these skin samples reveal sequences whose transcription was potentially altered in mammoth. We observe a tetradic structure for the inactive X chromosome in mammoth, distinct from the bipartite architecture seen in human and mouse. Generating chromosome-length genome assemblies for two other elephantids (Asian and African elephant), we find that the overall karyotype, and this tetradic Xi structure, are conserved throughout the clade. These results illustrate that cell-type specific epigenetic information can be preserved in ancient samples, in the form of DNA geometry, and that it may be feasible to perform de novo genome assembly of some extinct species.

genomics↗

bioRxiv2021

Fine-mapping of nuclear compartments using ultra-deep Hi-C shows that active promoter and enhancer elements localize in the active A compartment even when adjacent sequences do not

Megabase-scale intervals of active, gene-rich and inactive, gene-poor chromatin are known to segregate, forming the A and B compartments. Fine mapping of the contents of these A and B compartments has been hitherto impossible, owing to the extraordinary sequencing depths required to distinguish between the long-range contact patterns of individual loci, and to the computational complexity of the associated calculations. Here, we generate the largest published in situ Hi-C map to date, spanning 33 billion contacts. We also develop a computational method, dubbed PCA of Sparse, SUper Massive Matrices (POSSUMM), that is capable of efficiently calculating eigenvectors for sparse matrices with millions of rows and columns. Applying POSSUMM to our Hi-C dataset makes it possible to assign loci to the A and B compartment at 500 bp resolution. We find that loci frequently alternate between compartments as one moves along the contour of the genome, such that the median compartment interval is only 12.5 kb long. Contrary to the findings in coarse-resolution compartment profiles, we find that individual genes are not uniformly positioned in either the A compartment or the B compartment. Instead, essentially all (95%) active gene promoters localize in the A compartment, but the likelihood of localizing in the A compartment declines along the body of active genes, such that the transcriptional termini of long genes (>60 kb) tend to localize in the B compartment. Similarly, nearly all active enhancers elements (95%) localize in the A compartment, even when the flanking sequences are comprised entirely of inactive chromatin and localize in the B compartment. These results are consistent with a model in which DNA-bound regulatory complexes give rise to phase separation at the scale of individual DNA elements.

genomics↗

bioRxiv2021

Sex-specific multi-level 3D genome dynamics in the mouse brain

The female mammalian brain exhibits sex-hormone-driven plasticity during the reproductive period. Evidence implicates chromatin dynamics in gene regulation underlying this plasticity. However, whether ovarian hormones impact higher-order chromatin organization in post-mitotic neurons in vivo is unknown. Here, we mapped 3D genome of ventral hippocampal neurons across the estrous cycle and by sex in mice. In females, we found cycle-driven dynamism in 3D chromatin organization, including in estrogen-response-elements-enriched X-chromosome compartments, autosomal CTCF loops, and enhancer-promoter interactions. With rising estrogen levels, the female 3D genome becomes more similar to the male genome. Cyclical enhancer-promoter interactions are partially associated with gene expression and enriched for brain disorder-relevant genes. Our study reveals unique 3D genome dynamics in the female brain relevant to female-specific gene regulation, neuroplasticity, and disease risk.

neuroscience↗

bioRxiv2019

Functional characterization of RNase H1 proteins in Arabidopsis thaliana

ABSTRACTRNase H1 is an endonuclease specific towards RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. We show that Arabidopsis thaliana contains four RNase H1-like proteins originating from two gene duplication events and alternative splicing. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for activity. Two of those proteins are nuclear, one is localized to mitochondria and one to plastids. While the nuclear RNases H1 are dispensable, the presence of at least one organellar RNase H1 is required for embryonic development. The plastid protein RNH1C affects plastid DNA copy number and sensitivity to hydroxyurea. This indicates that three genomes present in each plant cell are served by at least one specialized RNase H1 protein.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2019

Immediate and deferred epigenomic signature of neuronal activation

Activity-driven transcription plays an important role in many brain processes, including those underlying memory and epilepsy. Here, we combine the genetic tagging of neuronal nuclei and ribosomes with various sequencing-based techniques to investigate the transcriptional and chromatin changes occurring at hippocampal excitatory neurons upon synchronous activation during status epilepticus and sparse activation during novel context exploration. The transcriptional burst, which affects both nucleus-resident non-coding RNAs and numerous protein-coding genes involved in neuroplasticity, is associated with a dramatic increase in chromatin accessibility of activity-regulated genes and enhancers, de novo binding of activity-regulated transcription factors, augmented promoter-enhancer interactions, and the formation of gene loops that bring together the TSS and TTS of strongly induced genes to sustain the fast re-loading of RNAPII complexes. Remarkably, some chromatin occupancy changes and interactions remain long after neuronal activation and may underlie the changes in neuronal responsiveness and circuit connectivity observed in these neuroplasticity paradigms.

neuroscience↗