Biology subjects

Markham, N. O.

Publications and source records attributed to Markham, N. O..

8 recordsLinked to original sources

bioRxiv2026

Aging restricts colorectal tumor growth by epigenetically silencing developmental gene programs

The incidence of early-onset colorectal cancer (CRC) has risen sharply in recent decades1, yet the biological basis underlying the distinct behavior of tumors arising in young versus aged tissues remains poorly understood. Here we show that aging reprograms the epigenetic landscape of the colon, restricting colon tumor growth through stable silencing of developmental and fetal gene programs. We find that colon tumors arising in aged mice are intrinsically less proliferative than those arising in young animals. Multi-omic profiling of normal colon and colon tumors reveals that aging drives DNA hypermethylation, loss of Polycomb-associated chromatin states, and reduced chromatin accessibility at a defined set of developmental genes that are bivalent (marked by both H3K27me3 and H3K4 methylation), transcriptionally active in colon tumors from young animals and repressed in both tumors and normal tissue from old animals. Among the genes most strongly repressed in old animals is Tacstd2 (Trop2), a regulator of fetal intestinal programs and epithelial stemness. Pharmacologic inhibition of DNA methylation reactivates the aging-silenced gene network in organoids from old animals, whereas genetic disruption of Tacstd2 suppresses growth and developmental transcriptional programs in young tumor organoids. TACSTD2, fetal gene signatures, and the aging-associated bivalent gene program are likewise repressed in late-onset vs. early-onset human colorectal cancers. Collectively, these findings identify age-associated epigenetic silencing of developmental gene programs as a causal mechanism that constrains colorectal tumor growth and provide a mechanistic framework for understanding the distinct biology of early-onset colorectal cancer.

cancer biology↗

bioRxiv2025

A targeted cell lysis mechanism facilitates toxin release in Clostridioides difficile

Clostridioides difficile infection depends on the production of two large toxins, TcdA and TcdB, encoded within a pathogenicity locus alongside the phage-like holin TcdE. The mechanism of toxin secretion remains actively debated, with current models proposing either TcdE-dependent non-lytic secretion or TcdE-independent lytic release. Here, we provide evidence for a unifying model where TcdE drives lysis in a phenotypically distinct subpopulation of cells. We show that TcdE, TcdA, and TcdB expression is restricted to a small fraction of cells exhibiting markers of active lysis and establish this subpopulation as the driver of severe pathogenic outcomes in a mouse model of CDI. Overexpression of TcdR, the sigma factor regulating the pathogenicity locus, triggers TcdE-dependent lysis, even in strains previously reported to employ TcdE-independent secretion mechanisms. Correlative light and electron microscopy combined with cryo-ET reveal a distinctive ultrastructure in lytic cells. Membrane vesicles accumulate between a disrupted inner membrane and intact peptidoglycan, alongside electron-dense material containing TcdA. These observations reveal a population-level strategy in which a minority of bacteria sacrifice themselves through TcdE-mediated lysis to release toxins as a 'bet-hedging strategy'.

microbiology↗

bioRxiv2025

Unbiased recording of clonal potency reveals species-specific regulation of mammalian intestine

The mammalian intestine regenerates rapidly after damage, yet the clonal dynamics and species-specific regulation of different populations remain poorly understood. Here we used synthetic or naturally occurring DNA alterations to reconstruct clonal histories of the mouse and human intestinal epithelium at single-cell resolution. In mice, we uncovered the clonal architecture of different cell types and their roles in regeneration, supporting a hierarchical regenerative response model. We identified a rare embryonic precursor population that persisted in the adult and was crucial for regeneration after irradiation. This population was marked by Tob2, which is required for nuclear transport of Ascl2. A parallel clonal analysis of 65 human colonic biopsies revealed secretory lineage bias and an age-associated decline in clonal diversity in the distal colon. Unlike highly proliferative murine Lgr5+ stem cells, human LGR5+ cells were found largely quiescent, revealing species-specific difference in clonal potency, and suggesting a distinct regulation of intestinal stemness.

genomics↗

bioRxiv2025

Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohns disease (CD) model (Tnf{Delta}ARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the hosts immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

cell biology↗

bioRxiv2024

Inhibition of EGFR/ErbB does not protect against C. difficile toxin B

Clostridioides difficile is a common cause of diarrhea and mortality, especially in immunosuppressed and hospitalized patients. C. difficile is a toxin-mediated disease, but the host cell receptors for C. difficile toxin B (TcdB) have only recently been revealed. Emerging data suggest TcdB interacts with receptor tyrosine kinases during infection. In particular, TcdB can elicit Epidermal Growth Factor Receptor (EGFR) transactivation in human colonic epithelial cells. The mechanisms for this function are not well understood, and the involvement of other receptors in the EGFR family of Erythroblastic Leukemia Viral Oncogene Homolog (ErbB) receptors remains unclear. Furthermore, in an siRNA-knockdown screen for protective genes involved with TcdB toxin pathogenesis, we show ErbB2 and ErbB3 loss resulted in increased cell viability. We hypothesize TcdB induces the transactivation of EGFR and/or ErbB receptors as a component of its cell-killing mechanism. Here, we show in vivo intrarectal instillation of TcdB in mice leads to phosphorylation of ErbB2 and ErbB3. However, immunohistochemical staining for phosphorylated ErbB2 and ErbB3 indicated no discernible difference between control and TcdB-treated mice for epithelial phospho-ErbB2 and phospho-ErbB3. Human colon cancer cell lines (HT29, Caco-2) exposed to TcdB were not protected by pre-treatment with lapatinib, an EGFR/ErbB2 inhibitor. Similarly, lapatinib pre-treatment failed to protect normal human colonoids from TcdB-induced cell death. Neutralizing antibodies against mouse EGFR failed to protect mice from TcdB intrarectal instillation as measured by edema, inflammatory infiltration, and epithelial injury. Our findings suggest TcdB-induced colonocyte cell death does not require EGFR/ErbB receptor tyrosine kinase activation.

microbiology↗

bioRxiv2021

Human colorectal pre-cancer atlas identifies distinct molecular programs underlying two major subclasses of pre-malignant tumors

Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SSLs). The origins and molecular landscapes of these histologically distinct pre-cancerous polyps remain incompletely understood. Here, we present an atlas at single-cell resolution of sporadic conventional tubular/tubulovillous ADs, SSLs, hyperplastic polyps (HPs), microsatellite stable (MSS) and unstable (MSI-H) CRC, and normal colonic mucosa. Using single-cell transcriptomics and multiplex imaging, we studied 69 datasets from 33 participants. We also examined separate sets of 66 and 274 polyps for RNA and targeted gene sequencing, respectively. We performed multiplex imaging on a tissue microarray of 14 ADs and 15 CRCs, and we integrated pre-cancer polyp data with published single-cell and The Cancer Genome Atlas (TCGA) bulk CRC data to establish potential polyp-cancer relationships. Striking differences were observed between ADs and SSLs that extended to MSS and MSI-H CRCs, respectively, reflecting their distinct origins and trajectories. ADs arose from WNT pathway dysregulation in stem cells, which aberrantly expanded and expressed a Hippo and ASCL2 regenerative program. In marked contrast, SSLs were depleted of stem cell-like populations and instead exhibited a program of gastric metaplasia in the setting of elevated cytotoxic inflammation. Using subtype-specific gene regulatory networks and shared genetic variant analysis, we implicated serrated polyps, including some HPs conventionally considered benign, as arising from a metaplastic program in committed absorptive cells. ADs and SSLs displayed distinct patterns of immune cell infiltration that may influence their natural history. Our multi-omic atlas provides novel insights into the malignant potential of colorectal polyps and serves as a framework for precision surveillance and prevention of sporadic CRC.

cancer biology↗

bioRxiv2020

Murine intrarectal instillation of purified recombinant C. difficile toxins enables mechanistic studies of pathogenesis

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota, et al. first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin (H&E) for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore-formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared with slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

microbiology↗

bioRxiv2020

Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multi-modal analyses of both microbes and host cells in a spatially-resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in Poloxamer-fixed samples. This in turn enabled us to integrate multi-modal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and crosslink at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

cell biology↗