Biology subjects

Stanford, W. L.

Publications and source records attributed to Stanford, W. L..

9 recordsLinked to original sources

bioRxiv2025

Spatiotemporal remodeling of chromatin topology architecture and H3K27me3 redistribution underlies vascular pathology in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder driven by the accumulation of the lamin A variant, progerin, leading to severe vascular pathology. While epigenetic alterations are implicated, the spatiotemporal reorganization of the higher-order chromatin and its functional impact on vascular smooth muscle cell (VSMC) transcription remain poorly defined. Through an integrated, longitudinal multi-omics approach combining in situ high-throughput chromosome conformation capture (Hi-C) and Cleavage Under Targets and Tagmentation (CUT&Tag) profiling of CTCF, SMC1A, H3K27me3, H3K27ac, and H3K36me3 with transcriptomic analyses from control and HGPS iPSC-derived VSMCs, we reveal that global topologically associating domain (TAD) architecture remains largely intact in HGPS. However, the internal chromatin states of TADs undergo dynamic, passage-specific remodeling, characterized by a progressive accumulation of broad H3K27me3-repressed domains. This is accompanied by a loss of A/B compartment segregation, as confirmed by DNA-FISH, and a pronounced depletion of interchromosomal intermingling, specifically surrounding downregulated genes, suggesting that these interchromosomal contacts are essential for maintaining transcriptional competence in normal VSMCs. Crucially, we uncover widespread rewiring of enhancer-promoter (E-P) loops, which is linked to the dysregulation of genes critical for vascular development, extracellular matrix organization, and atherosclerosis. Our study demonstrates that spatiotemporal redistribution of repressive histone marks and reorganization of E-P interactions within a structurally resilient TAD framework underpin widespread transcriptional dysregulation in HGPS vascular pathogenesis. This uncovers a critical dissociation between higher-order chromatin architecture and histone modification landscape, providing a mechanistic basis for the failure of vascular homeostasis in progeria.

genomics↗

bioRxiv2025

HDAC2 inhibition restores H4K16 Acetylation and Rescues Cellular Senescence in Hutchinson-Gilford progeria syndrome

Histone H4 lysine 16 acetylation (H4K16ac) controls chromatin compaction, replication-coupled chromatin maturation and double-strand break repair. It is also depleted in Hutchinson-Gilford progeria syndrome (HGPS) vascular smooth muscle cells (VSMCs). To identify the enzymes that set steady-state H4K16ac, we depleted KAT5, KAT8, all eleven zinc-dependent HDACs and all seven sirtuins in HeLa and U2OS cells, then in unaffected control and HGPS VSMCs. KAT8 depletion markedly reduced H4K16ac; KAT5 depletion had little effect. Among erasers, HDAC2 was dominant: its depletion or overexpression bidirectionally modulated H4K16ac, HDAC1 did neither, and SIRT1 depletion raised H4K16ac in these lines but far less in HGPS VSMCs. HDAC2-directed inhibitors (BRD4884, MI192, Santacruzamate A) restored H4K16ac in HGPS VSMCs, whereas the SIRT1 inhibitor EX527 did not. They also improved nuclear architecture, reduced {gamma}H2AX signaling, preserved Ki67 positivity across serial passage, and limited senescence-associated {beta}-galactosidase accumulation. HDAC2 is therefore the predominant class I regulator of steady-state H4K16ac and a candidate target for limiting senescence in HGPS VSMCs.

molecular biology↗

bioRxiv2024

Loss of VHL-mediated pRb regulation promotes clear cell renal cell carcinoma

The von Hippel-Lindau (VHL) tumor suppressor is a component of E3 ubiquitin ligase complexes that target cellular substrates for proteasome-mediated degradation. VHL inactivation by genetic aberrations is observed in most sporadic cases of clear cell renal cell carcinoma (ccRCC). VHL loss leads to constitutive stabilization of E3 ligase targets, including hypoxia inducible factor (HIF), in VHL-associated tumors. HIF stabilization upon VHL loss promotes transactivation of hypoxia responsive genes, which contributes to ccRCC development. However, several HIF-independent VHL targets have also been implicated in the promotion of tumorigenesis. Using proximity labeling to identify proteasomal VHL interactors, we identified retinoblastoma protein (pRb) as a novel substrate of VHL. Mechanistically, VHL interacts with pRb in an oxygen-sensitive manner, promoting its ubiquitin-mediated degradation. Concordantly, VHL-inactivation results in pRb hyperstabilization. Functionally, the hyperstabilization of pRb in ccRCC promoted tumorigenesis in vitro and in mouse models. We also show that downstream transcriptional changes induced by pRb hyperstabilization may contribute to ccRCC tumor development. Together, our findings reveal a novel VHL-related pathway which can be therapeutically targeted to inhibit ccRCC tumor development.

cancer biology↗

bioRxiv2023

Differential and lasting gene expression changes in circulating CD8 T cells in chronic HCV infection with cirrhosis and related insights on the role of Hedgehog signaling

The impact of chronic hepatic infection on antigen non-specific immune cells in circulation is not well understood and may influence long term health. We reported lasting global hyperfunction of circulating CD8 T cells in HCV-infected individuals with cirrhosis. Whether gene expression patterns in bulk CD8 T cells are associated with the severity of liver fibrosis in HCV infection is not known. RNA sequencing of blood CD8 T cells from treatment-naive, HCV-infected individuals with minimal (Metavir F0-1 [≤] 7.0 kPa) or advanced fibrosis or cirrhosis (F4 [≥] 12.5 kPa), before and after direct-acting antiviral therapy, was performed. Principal component analyses determined robust differences in over 350 genes expressed by CD8 T cells from HCV-infected individuals with minimal or advanced fibrosis and data suggests this remains relatively stable after viral clearance. Gene ontology analyses identified disaggregated gene expression related to cellular metabolism, including upregulated phospholipase, phosphatidyl-choline/inositol activity and second-messenger-mediated signaling, while genes in pathways associated with nuclear processes, RNA transport and cytoskeletal dynamics were reduced. Gene Set Enrichment Analysis identified decreased expression of genes regulated by the cMyc and E2f transcription factors in cirrhotics, compared to the minimal fibrosis group, as well as reduced expression of genes linked to oxidative phosphorylation, mTOR signaling, and more. Upregulated gene sets in cirrhotics included IFN-, -{gamma}, TGF-{beta} response genes, apoptosis and apical surface pathways, among others. The hedgehog (Hh) signaling pathway was the top featured gene set upregulated in cirrhotics. Inhibition of Hh signaling with cyclopamine ablated CD8 T cell IFN-{gamma} production, suggesting its involvement in hyperfunction. This is the first analysis of bulk CD8 T cell gene expression profiles in HCV infection in the context of liver fibrosis severity, and suggests cirrhosis significantly reprograms the CD8 T cell pool. The novel finding of increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease.

immunology↗

bioRxiv2022

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy

Parkinsonas disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of -Synuclein (-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the -Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress -Syn replicate several features of PD. Decreasing total -Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of -Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of -Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the -Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical -Syn pathology, indicated by reduced numbers of pS129 -Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic -Syn species. We further validated these findings on pS129 -Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated -Syn in human neurons, in PFF- challenged rat neurons and in the brains of -Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

neuroscience↗

bioRxiv2022

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

microbiology↗

bioRxiv2021

Three-dimensional drug screen identifies HDAC inhibitors as therapeutic agents in mTORC1-driven lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring constitutive mTORC1 signaling. Rapamycin is the only clinically approved disease-modifying treatment, but its action is cytostatic and disease progresses upon its withdrawal. There is a critical need to identify novel agents that prevent the invasive phenotype and/or eradicate the neoplastic LAM cells. Here, we employed novel cellular and extracellular models to screen for candidate therapeutics in a physiologically relevant setting. We observed that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional tissue culture plastic. Leveraging our culture system, we conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. We identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. Unexpectedly, we observed that next generation ATP-competitive mTORC1/2 inhibitors potentiate invasion. We determined anti-invasive effects of HDAC inhibitors to be independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Drug performance was subsequently evaluated at the single cell level in zebrafish xenografts. We observed consistent therapeutic efficacy in vivo at equivalent concentrations to those used in vitro, substantiating HDAC inhibitors as potential therapeutic candidates for pursuit in patients with LAM. One Sentence SummaryWe performed a drug screen in 3D and discovered HDAC inhibitors exhibit therapeutic efficacy in models of the lung disease lymphangioleiomyomatosis.

cell biology↗

bioRxiv2019

TSC2-deficiency potentiates a catabolic signaling switch that differentiates neural and neural crest lineage development and progressive disease manifestations

Lymphangioleiomyomatosis (LAM) is a rare lung disease marked by cystic destruction caused by invasive LAM cells harboring loss-of-function mutations in TSC2 that exhibit dysregulated mTORC1 signaling. Rapamycin, the only approved treatment, is not curative. Therapeutic discovery has been limited by inadequate cell and animal models due to the unknown LAM cell of origin. We report a novel TSC2--/-- human pluripotent stem cell (hPSC)-derived neural crest cell (NCC) model, which replicates key LAM features. Leveraging this model, temporal RNA sequencing and genome-wide CRISPR knockout screens identified synthetic lethal genes including TWIST1 and SUN4. Antisense oligonucleotides (ASOs) targeting TWIST1 and SUN4 demonstrated selective TSC2 mutant NCC cytotoxicity and inhibited invasion. Using a "test-withdraw-monitor" clinical trial paradigm, TWIST1 and SUN4 ASOs profoundly suppressed tumor viability in a Tsc2--/-- mouse allograft model and eradicated 40% of tumors, outperforming standard of care rapamycin and highlighting their promise as novel cytoablative LAM therapeutics.

cancer biology↗

bioRxiv2019

Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

bioengineering↗