Biology subjects

Garfield, D.

Publications and source records attributed to Garfield, D..

7 recordsLinked to original sources

bioRxiv2026

Deep learning design and in vivo validation of Müller glia-specific cis-regulatory elements

Effective recombinant adeno-associated virus gene therapies require promoters that are compact and cell-type-specific. Ideally, promoters should also exhibit functional conservation when tested in model organisms to ensure that preclinical findings translate reliably to human patients. Here, we introduce a deep learning framework for designing cis-regulatory elements (CREs) meeting these criteria, applied to retinal Muller glia (MG). Using single-cell chromatin accessibility data from human and mouse retinas, we trained species-specific models to predict cell-type accessibility, and designed compact CREs using two complementary strategies. In silico validation predicted that the designed CREs exhibit high MG-specific accessibility (on-target) in both species with minimal off-target accessibility across hundreds of human cell types and tissues. Mechanistic analysis revealed that the predicted MG accessibility is driven by the creation of LHX2 motifs. In vivo validation confirmed that the designed CREs successfully restrict reporter expression to MG in the murine retina. Our deep learning framework is highly generalizable and enables the rapid design of compact, on-target, and species-conserved CREs for precision gene therapy.

genomics↗

bioRxiv2026

NINTEDANIB AND PIRFENIDONE AFFECT GROWTH AND DIFFERENTIATION OF HUMAN ALVEOLAR TYPE 2 CELLS

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterized by epithelial cell senescence. Pirfenidone and nintedanib are approved drugs for the treatment of IPF. They significantly slow disease progression, but their mechanisms of action, especially on alveolar type 2 (AT2) cells, are poorly understood. We addressed this question by evaluating colony formation and growth of human AT2 cells co-cultured with fibroblasts in organoid culture in the presence of pirfenidone and nintedanib. We further evaluated molecular changes induced by these drugs via single cell RNA-seq of treated organoids. MethodsAT2 cells isolated from normal donor lungs or IPF patients were mixed with human fibroblasts in 3D culture and grown in the absence or presence of pirfenidone or nintedanib. After 14 days in culture, the organoids were quantified and cells extracted from Matrigel for single cell RNA-seq. ResultsAT2 cell organoids cultured in the presence of pirfenidone or nintedanib resulted in increased colony formation and, in the case of nintedanib, in larger colonies. We observed that untreated or pirfenidone treated AT2 cells lost surfactant protein C (SFTPC) expression and acquired an expression profile consistent with keratin (KRT)17high/KRT5- basaloid cells, whereas a larger proportion of nintedanib treated cells retained SFTPC expression. In contrast, AT2 cells treated with TGF{beta} inhibitor exhibited intermediate (SFTPC-/KRT17low) gene expression profile. ConclusionThese results suggest that nintedanib maintains an AT2-like expression state in culture and acts proximal to TGF{beta}. Conflict of Interest StatementPJW was supported by grants from Boehringer Ingelheim, Roche, Sanofi, Pliant and Arda Therapeutics and received personal fees from Boehringer Ingelheim and Sanofi. None of these companies had a role in the design or analysis of the study or in the writing of the manuscript. ASM, GP, JRR and DG are employees of Genetech. The other authors have no conflicts of interest to declare.

cell biology↗

bioRxiv2025

The Establishment of Cell-Type Specific Gene Regulation in the Sea Urchin Embryo

Cell-fate commitment in metazoan development relies on precise gene regulatory programs. This study presents a comprehensive single-cell atlas of gene expression (scRNA-seq), nascent transcription (scSLAM-seq), and chromatin accessibility (scATAC-seq) in the purple sea urchin, Strongylocentrotus purpuratus, from early cleavage to pluteus larva stages. Our findings reveal a dynamic regulatory landscape with extensive usage of distal and intronic regulatory elements, which often exhibit cell-type-specific motifs and accessibility profiles that closely track gene expression. We identify a major wave of zygotic genome activation (ZGA) at the 128-cell stage, coinciding with the loss of developmental plasticity, alongside evidence of restricted, lineage-specific gene activation preceding widespread ZGA. Motif analysis highlights distinct regulatory grammars for these early accessible regions. Regulatory element usage largely clusters by germ layer, indicating shared accessibility among related cell types. We delve into the regulatory intricacies of neurons and skeletogenic cells. Sea urchin neurodevelopment proceeds through three distinct lineages, utilizing transcription factors with conserved roles in mammalian neurogenesis. Surprisingly, skeletogenic cells show significant transcriptional and regulatory diversity across their subpopulations, and we identify novel genes associated with calcification. This research offers unprecedented insights into the dynamic regulatory genome of a non-chordate deuterostome, highlighting both conserved principles of gene regulation and unique features that underscore the sea urchins importance as a model for understanding developmental and evolutionary genomics in ecologically critical marine species.

developmental biology↗

bioRxiv2025

Maladaptive inflammatory signaling in old mice impairs colonic regeneration by promoting a sustained fetal-like epithelial state

Aging is associated with a decline in the regenerative capacity of many tissues. Central to this decline is a complex interplay between inflammation and stem cell function. How these two processes are linked and influence regenerative capacity remains unclear. Here, we undertake a comprehensive assessment of age-related changes in the mouse colon at single-cell resolution. A survey of immune and epithelial compartments revealed a hyperactivated inflammatory state in the colon of old mice characterized by the induction of an interferon {gamma} (IFN{gamma}) response signature in immune cells. This does not result in increased inflammation under homeostasis, but triggers a disproportionate inflammatory response, disrupting regeneration after challenge with the enteropathogen Citrobacter rodentium. Colons of old mice exhibit higher production of IFN{gamma} by T and innate lymphoid cells (ILCs) that are associated with reduced Lgr5+ stem cells and decreased epithelial proliferation. Interestingly, we find aged intestinal epithelial cells to be hypersensitive to IFN{gamma} signaling, inducing a regeneration-associated fetal-like gene expression signature that, in turn, renders these cells more sensitive to IFN{gamma}-induced apoptosis. Our findings reveal an age-related imbalance in the interaction between the immune and epithelial compartments in the colon, priming the system for excessive inflammatory responses and the emergence of a hypersensitive epithelial cell state thus derailing proper repair of the intestinal epithelium after injury.

immunology↗

bioRxiv2024

Decoding sequence determinants of gene expression in diverse cellular and disease states

Sequence-to-function models that predict gene expression from genomic DNA sequence have proven valuable for many biological tasks, including understanding cis-regulatory syntax and interpreting non-coding genetic variants. However, current state-of-the-art models have been trained largely on bulk expression profiles from healthy tissues or cell lines, and have not learned the properties of precise cell types and states that are captured in large-scale single-cell transcriptomic datasets. Thus, they lack the ability to perform these tasks at the resolution of specific cell types or states across diverse tissue and disease contexts. To address this gap, we present Decima, a model that predicts the cell type- and condition- specific expression of a gene from its surrounding DNA sequence. Decima is trained on single-cell or single-nucleus RNA sequencing data from over 22 million cells, and successfully predicts the cell type-specific expression of unseen genes based on their sequence alone. Here, we demonstrate Decimas ability to reveal the cis-regulatory mechanisms driving cell type-specific gene expression and its changes in disease, to predict non-coding variant effects at cell type resolution, and to design regulatory DNA elements with precisely tuned, context-specific functions.

genomics↗

bioRxiv2024

regLM: Designing realistic regulatory DNA with autoregressive language models

Cis-regulatory elements (CREs), such as promoters and en-hancers, are DNA sequences that regulate the expression of genes. The activity of a CRE is influenced by the order, composition and spacing of sequence motifs that bind to proteins called transcription factors (TFs). Synthetic CREs with specific properties are needed for biomanufacturing as well as for many therapeutic applications including cell and gene therapy. Here, we present regLM, a framework to design synthetic CREs with desired properties, such as high, low or cell type-specific activity, using autoregressive language models in conjunction with supervised sequence-to-function models. We used our framework to design synthetic yeast promoters and cell type-specific human enhancers. We demonstrate that the synthetic CREs generated by our approach are not only predicted to have the desired functionality but also contain biological features similar to experimentally validated CREs. regLM thus facilitates the design of realistic regulatory DNA elements while providing insights into the cis-regulatory code.

genomics↗

bioRxiv2020

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in wildtype and cloche/npas4l mutant zebrafish embryos

DNA accessibility of cis regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many of the genes that regulate embryonic development have been described, the underlying CRE dynamics controlling their expression remain largely unknown. To address this, we applied single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq) to whole 24 hours post fertilization (hpf) stage zebrafish embryos and developed a new computational tool, ScregSeg, that selects informative genome segments and classifies complex accessibility dynamics. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization, expanding knowledge of regulatory principles between chromatin modalities. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos revealed novel cellular roles for this hemato-vascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work constitutes a valuable resource for future studies in developmental, molecular, and computational biology.

genomics↗