Biology subjects

Gracz, A. D.

Publications and source records attributed to Gracz, A. D..

9 recordsLinked to original sources

bioRxiv2026

Chromatin State Distinguishes Injury-Responsive from State-Stabilizing Transcriptional Programs in Hybrid Hepatocytes

Liver injury induces rapid transcriptional responses in hepatocytes, yet the chromatin features that distinguish injured hepatocytes from healthy hepatocytes remain poorly understood. Using an integrated functional genomics approach combining bulk RNA-seq, ATAC-seq, and CUT&Tag profiling of H3K27ac and H3K27me3, we define the transcriptional and chromatin landscape of Sox9-expressing hepatocytes, which exhibit gene expression consistent with both hepatocyte and biliary identity. Under homeostatic conditions, Sox9+ hybrid hepatocytes (HybHeps) are rare and confined to the periportal space, while chronic injury induces an expansion of Sox9+ metaplastic hepatocytes (MetHeps). We identify three classes of differentially expressed genes associated with injury-responsive, state-associated, or shared regulatory programs and demonstrate that these classes are governed by distinct chromatin mechanisms. Injury-responsive transcription is driven primarily by dynamic chromatin accessibility remodeling at NF-{kappa}B- and AP-1-enriched regulatory elements, while state-associated and shared programs are reinforced through selective H3K27ac and H3K27me3 modification with comparatively stable accessibility. Relative to conventional hepatocytes, HybHeps encode a permissive chromatin landscape at injury-responsive loci under homeostatic conditions, consistent with epigenetic priming that facilitates rapid inflammatory activation. Projection of mouse-derived gene programs onto a human liver single-cell atlas encompassing both healthy and diseased hepatocytes confirms that SOX9-expressing hepatocytes preferentially engage injury-associated inflammatory modules while attenuating hepatocyte metabolic identity programs. Together, these findings define a chromatin-based regulatory dichotomy between inflammatory responsiveness and hybrid hepatocyte cell state stability, providing mechanistic insight into how differentiated epithelial cells integrate inflammatory signals while preserving cell state.

developmental biology↗

bioRxiv2026

Multi-gene biomarkers reveal spatial organization and subpopulation-specific damage response in intrahepatic biliary epithelial cells

Background & AimsIntrahepatic biliary epithelial cell (BEC) heterogeneity remains challenging to define. Here, we sought to identify BEC subpopulations and biomarkers in mouse liver. MethodsWe performed scRNA-seq on Sox9EGFP+ liver epithelium from mice subjected to bile duct ligation (BDL) and sham controls. A machine learning algorithm, NSForest, identified minimal, multi-gene signatures for BEC subpopulations. These "metagenes" were validated using hybridization chain reaction (HCR) FISH in tissue sections from wild-type mice and on primary BECs expanded in vitro. Metagenes were used to match BDL subpopulations to their corresponding sham subpopulations for differential gene expression (DGE) analysis. ResultsWe identified 4 BEC subpopulations in sham controls, each associated with 1-2 gene metagenes. Spatial localization of metagene-defined BEC subpopulations by HCR FISH revealed heterogeneous cellular composition of intrahepatic bile ducts. BECs belonging to a given subpopulation were most likely to have neighbors of the same identity, forming homogenous cellular compartments within ducts. BDL downregulated subpopulation-specific genes and upregulated a damage-associated gene set. BDL samples also included a proliferative subpopulation not found in sham controls, which contained populations enriched for three of the four metagenes. All BEC subpopulations were also found in monolayers in vitro, where they clustered spatially with BECs of the same subtype. ConclusionsNovel metagene biomarkers of BEC subpopulations facilitated spatial localization of BECs in situ, identified subpopulation specific injury responses, and confirmed that BEC heterogeneity is preserved in vitro. The presence of locally homogenous BEC "neighborhoods" in vitro suggests some degree of BEC organization may be epithelial-autonomous.

cell biology↗

bioRxiv2025

Tet1 safeguards lineage allocation in intestinal stem cells

Intestinal stem cells (ISCs) balance self-renewal and differentiation to maintain the intestinal epithelial barrier, which is replaced weekly throughout adult life. Genetic control of ISC differentiation is well-defined relative to transcription factor (TF) activity, but less is known regarding the role of chromatin regulation in ISC biology. Prior work from our lab and others has shown that Tet1, a chromatin modifying enzyme involved in DNA demethylation, is specifically enriched in ISCs and early secretory progenitors. While constitutive loss of Tet1 is associated with defects in early postnatal ISC development, its role in adult ISC biology remains unknown. Here, we show that Tet1 safeguards ISC fate decisions by reducing sensitivity to extrinsic signaling. Inducible, intestine-specific Tet1 knockout mice (Tet1iKO) exhibit environmentally sensitive phenotypes, including absorptive differentiation bias and premature expression of mature absorptive transcripts in ISCs. These phenotypes are largely "silenced" in animals housed in a high-level barrier facility, where Tet1iKO epithelium closely resembles controls. Despite the lack of baseline phenotype in these conditions, Tet1iKO mice retain increased sensitivity to pro-differentiation signaling. In vivo, succinate administration induces increased tuft and goblet cell hyperplasia in the absence of Tet1, while Tet1iKO organoids cultured with IL-4 or DAPT exhibit increased tuft and enteroendocrine cell specification, respectively. While ATAC-seq of Tet1iKO ISCs reveals minimal changes in chromatin accessibility, footprinting analysis suggests increased binding of lineage-specific TFs and CTCF even in the absence of cellular phenotypes. Together, our data demonstrate that Tet1 serves as a "buffer" against ISC differentiation and suggest that it does so in a lineage agnostic manner that is not dependent on changes in chromatin accessibility.

cell biology↗

bioRxiv2025

Quantitative 3D imaging of mouse and human intrahepatic bile ducts in homeostasis and liver injury

Intrahepatic bile ducts (IHBDs) form a complex hierarchical network essential for liver function. Remodeling and expansion of this network during ductular reaction (DR) is a hallmark of liver disease that can be a key indicator of disease severity. Conventional histology fails to capture the full extent of IHBD structural changes following injury due to the complex 3D organization of the IHBD network which limits understanding of DR, especially in human tissue. A major barrier to leveraging 3D imaging as a diagnostic tool is the absence of standardized pipelines for IHBD imaging and analysis. Here, we establish a robust 3D IHBD imaging and analysis workflow and apply it to both mouse and human liver tissues. This pipeline enables quantification of tissue and individual duct ("segment") level features and identifies features of invasive and noninvasive DR. In mouse models, we uncover regional phenotypes, including IHBD diverticula following duct blockage and the formation of anastomosed clusters after hepatocellular injury. Finally, we apply our 3D imaging and analysis workflow to quantify IHBD networks in human liver tissue. This work deepens our understanding of IHBD architecture in homeostasis and injury and lays the groundwork for advanced phenotyping of IHBD morphologies in mice and humans with relevance to next-generation experimental and diagnostic approaches to liver disease.

pathology↗

bioRxiv2024

Tritrichomonas muris sensitizes the intestinal epithelium to doxorubicin-induced apoptosis

Doxorubicin (DXR) is a widely used chemotherapy drug that can induce severe intestinal mucositis. While the influence of gut bacteria on DXR-induced damage has been documented, the role of eukaryotic commensals remains unexplored. We discovered Tritrichomonas muris (Tmu) in one of our mouse colonies exhibiting abnormal tuft cell hyperplasia, prompting an investigation into its impact on DXR-induced intestinal injury. Mice from Tmu-colonized and Tmu-excluded facilities were injected with DXR, and tissue morphology and gene expression were evaluated at acute injury (6 h) and peak regeneration (120 h) phases. Contrary to previous reports, DXR did not significantly alter villus height, crypt depth, or crypt density in any mice. However, we did observe apoptosis, measured by cleaved caspase 3 (CC3) staining, in intestinal crypts at 6 h post-DXR that was significantly higher in mice colonized by Tmu. Interestingly, while DXR did not alter the expression of active and facultative intestinal stem cell (ISC) marker genes in control mice, it significantly reduced their expression in Tmu+ mice. Tmu, but not DXR, is also associated with increased inflammation and expression of the type 2 cytokines IL-5 and IL-13. However, pre-treatment of intestinal organoids with these cytokines is not sufficient to drive elevated DXR-induced apoptosis. These findings highlight the significant influence of commensal microbiota, particularly eukaryotic organisms like Tmu, on intestinal biology and response to chemotherapy, underscoring the complexity of gut microbiota interactions in drug-induced mucositis. NEW & NOTEWORTHYOur study found that the eukaryotic commensal Tritrichomonas muris (Tmu) significantly increases DXR-induced intestinal apoptosis in mice, despite no changes in tissue morphology. Tmu also reduces intestinal stem cell gene expression post-DXR injury, and elevates inflammation and type 2 cytokine expression in the absence of injury. In vitro organoid assays suggest that type 2 cytokines alone are insufficient to promote increased DXR-associated apoptosis. These findings emphasize the complex role of gut microbiota in drug-induced intestinal damage.

cell biology↗

bioRxiv2024

Lgr5+ intestinal stem cells are required for organoid survival after genotoxic injury

Progenitors and mature cells can maintain the intestinal epithelium by dedifferentiation and facultative intestinal stem cell (fISC) function when active ISCs (aISCs) are lost to damage. Here, we sought to model fISC activation in intestinal organoids with doxorubicin (DXR), a chemotherapeutic known to ablate Lgr5+ aISCs in vivo. We identified low and high doses of DXR compatible with long-term organoid survival. Similar fISC gene activation was observed between organoids treated with low vs high DXR, despite significantly decreased survival at the higher dose. aISCs exhibit dose-dependent loss after DXR but survive at doses compatible with organoid survival. We ablated residual aISCs after DXR using a Lgr52A-DTR allele and observed that aISC survival of the initial genotoxic insult is required for organoid survival following DXR. These results suggest that while typical fISC genes are activated by DXR injury in organoids, functional stemness remains dependent on the aISC pool. Our data establish a reproducible model of DXR injury in intestinal organoids and reveal differences in in vitro responses to an established in vivo damage modality.

cell biology↗

bioRxiv2024

Sox9 links biliary maturation to branching morphogenesis

Branching morphogenesis couples cellular differentiation with development of tissue architecture. Intrahepatic bile duct (IHBD) morphogenesis is initiated with biliary epithelial cell (BEC) specification and eventually forms a heterogeneous network of large ducts and small ductules. Here, we show that Sox9 is required for developmental establishment of small ductules. IHBDs emerge as a webbed structure by E15.5 and undergo morphological maturation through 2 weeks of age. Developmental knockout of Sox9 leads to decreased postnatal branching morphogenesis, manifesting as loss of ductules in adult livers. In the absence of Sox9, BECs fail to mature and exhibit elevated TGF-{beta} signaling and Activin A. Activin A induces developmental gene expression and morphological defects in BEC organoids and represses ductule formation in postnatal livers. Our data demonstrate that adult IHBD morphology and BEC maturation is regulated by the Sox9-dependent formation of precursors to ductules during development, mediated in part by downregulation of Activin A.

developmental biology↗

bioRxiv2020

Sox9EGFP defines biliary epithelial heterogeneity downstream of Yap activity

Intrahepatic bile ducts are lined by biliary epithelial cells (BECs). However, defining the genetic heterogeneity of BECs remains challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize Sox9EGFP transgene expression in the liver and demonstrate that GFP expression levels are associated with distinct cell types. BECs express "low" or "high" levels of GFP, while periportal hepatocytes express "sublow" GFP. Sox9EGFP distribution varies by duct size, with GFPhigh BECs found at greater numbers in smaller ducts. RNA-seq reveals distinct gene expression signatures for Sox9EGFP populations and enrichment of Notch and Yap signaling in GFPlow and GFPhigh BECs. All GFP+ populations are capable of forming organoids, but demonstrate interpopulation differences in organoid survival and size, dependent on media conditions. Organoids derived from Sox9EGFP populations also demonstrate differential activation of HNF4A protein in hepatocyte media conditions, suggesting variable potency in BEC subpopulations. We find that Yap signaling is required to maintain Sox9 expression in biliary organoids, and that bile acids are insufficient to induce Yap activity or Sox9 in vivo and in vitro. Our data demonstrate that Sox9EGFP levels provide a readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

cell biology↗

bioRxiv2019

Intestinal stem cell differentiation is associated with dynamic changes in the chromatin landscape

Intestinal stem cell (ISC) plasticity is thought to be regulated by broadly-permissive chromatin shared between ISCs and their progeny. Here, we utilize a Sox9EGFP reporter to examine chromatin across ISC differentiation. We find that open chromatin regions (OCRs) can be defined as broadly-permissive or dynamic in a locus-specific manner, with dynamic OCRs found primarily in loci consistent with distal enhancers. By integrating gene expression with chromatin accessibility at transcription factor (TF) motifs in context of Sox9EGFP populations, we classify broadly-permissive and dynamic chromatin relative to TF usage. These analyses identify known and potential regulators of ISC differentiation via their association with dynamic changes in chromatin. We observe ISC expansion in Id3-null mice, consistent with computational predictions. Finally, we examine the relationship between gene expression and 5-hydroxymethylcytosine (5hmC) in Sox9EGFP populations, which reveals 5hmC enrichment in absorptive lineage specific genes. Our data demonstrate that intestinal chromatin dynamics can be quantitatively defined in a locus-specific manner, identify novel potential regulators of ISC differentiation, and provide a chromatin roadmap for further dissecting the role of cis regulation of cell fate in the intestine.

genomics↗