Biology subjects

Fields, A. J.

Publications and source records attributed to Fields, A. J..

6 recordsLinked to original sources

bioRxiv2026

Epigenetic progression of pancreatic cancer to aggressive subtypes involves alternate routes of lineage reprogramming in subtype-intermediate progenitor cells

Pancreatic ductal adenocarcinoma (PDAC) progression involves malignant cell state plasticity. Epigenetic changes underlie this plasticity, yet the PDAC cis-regulatory landscape remains understudied. To address this, we profiled 33 primary tumors and 7 metastases from 39 patients with single-cell ATAC-seq, paired with 10 single-cell RNA-seq profiles. We found that epigenetic GATA6+/KRT17+ co-accessibility identifies a classical-basal subtype-intermediate progenitor state (SIP) associated with better clinical outcomes. SIP cells display limited epigenetic reprogramming from premalignant epithelium and retain gastric-intestinal differentiation reminiscent of neoplastic precursors. Lineages without GATA6+/KRT17+ co-accessibility exhibit greater lineage and epithelial-mesenchymal plasticity. Classical PDACs that repress basal gene accessibility activate neural-like progenitor (NRP) and tuft lineage enhancers, whereas basal committed tumors display esophageal transdifferentiation. Compared to SIP, classical-NRP and basal committed tumors have poorer outcomes, and show distinct PD-1/PD-L1 immune proteomic phenotypes and prognostic myofibroblast epigenetic states, respectively. Our work reveals links between lineage reprogramming, EMT, and epigenetic progression in human PDAC.

cancer biology↗

bioRxiv2019

Spatially-mapped single-cell chromatin accessibility

High-throughput single-cell epigenomic assays can resolve the heterogeneity of cell types and states in complex tissues, however, spatial orientation within the network of interconnected cells is lost. Here, we present a novel method for highly scalable, spatially resolved, single-cell profiling of chromatin states. We use high-density multiregional sampling to perform single-cell combinatorial indexing on Microbiopsies Assigned to Positions for the Assay for Transposase Accessible Chromatin (sciMAP-ATAC) to produce single-cell data of an equivalent quality to non-spatially resolved single-cell ATAC-seq, where each cell is localized to a three-dimensional position within the tissue. A typical experiment comprises between 96 and 384 spatially mapped tissue positions, each producing 10s to over 100 individual single-cell ATAC-seq profiles, and a typical resolution of 214 cubic microns; with the ability to tune the resolution and cell throughput to suit each target application. We apply sciMAP-ATAC to the adult mouse primary somatosensory cortex, where we profile cortical lamination and demonstrate the ability to analyze data from a single tissue position or compare a single cell type in adjacent positions. We also profile the human primary visual cortex, where we produce spatial trajectories through the cortex. Finally, we characterize the spatially progressive nature of cerebral ischemic infarct in the mouse brain using a model of transient middle cerebral artery occlusion. We leverage the spatial information to identify novel and known transcription factor activities that vary by proximity to the ischemic infarction core with cell type specificity.

genetics↗

bioRxiv2019

Improved single-cell ATAC-seq reveals chromatin dynamics of in vitro corticogenesis

Development is a complex process that requires the precise modulation of regulatory gene networks controlled through dynamic changes in the epigenome. Single-cell-omic technologies provide an avenue for understanding the mechanisms of these processes by capturing the progression of epigenetic cell states during the course of cellular differentiation using in vitro or in vivo models1. However, current single-cell epigenomic methods are limited in the information garnered per individual cell, which in turn limits their ability to measure chromatin dynamics and state shifts. Single-cell combinatorial indexing (sci-) has been applied as a strategy for identifying single-cell-omic originating libraries and removes the necessity of single-cell, single-compartment chemistry2. Here, we report an improved sci-assay for transposase accessible chromatin by sequencing (ATAC-seq), which utilizes the small molecule inhibitor Pitstop 2 (scip-ATAC-seq)3. We demonstrate that these improvements, which theoretically could be applied to any in situ transposition method for single-cell library preparation, significantly increase the ability of transposase to enter the nucleus and generate highly complex single-cell libraries, without altering biological signal. We applied sci-ATAC-seq and scip-ATAC-seq to characterize the chromatin dynamics of developing forebrain-like organoids, an in vitro model of human corticogenesis4. Using these data, we characterized novel putative regulatory elements, compared the epigenome of the organoid model to human cortex data, generated a high-resolution pseudotemporal map of chromatin accessibility through differentiation, and measured epigenomic changes coinciding with a neurogenic fate decision point. Finally, we combined transcription factor motif accessibility with gene activity (GA) scores to directly observe the dynamics of complex regulatory programs that regulate neurogenesis through developmental pseudotime. Overall, scip-ATAC-seq increases information content per cell and bolsters the potential for future single-cell studies into complex developmental processes.

genomics↗

bioRxiv2019

Suppressed Osteocyte Perilacunar / Canalicular RemodelingPlays a Causal Role in Osteoarthritis

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. To determine if PLR suppression plays a causal role in OA, we ablated the PLR enzyme MMP13 in osteocytes, while leaving chondrocytic MMP13 intact. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, this osteocyte-intrinsic PLR defect was sufficient to reduce cartilage proteoglycan content and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, osteocyte PLR is a critical regulator of cartilage homeostasis. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and identify the causal role of suppressed perilacunar/canalicular remodeling in osteoarthritis.

physiology↗

bioRxiv2018

Developmental Mechanisms Linking Form and Function During Jaw Evolution

How does form arise during development and change during evolution? How does form relate to function, and what enables embryonic structures to presage their later use in adults? To address these questions, we leverage the distinct functional morphology of the jaw in duck, chick, and quail. In connection with their specialized mode of feeding, duck develop a secondary cartilage at the tendon insertion of their jaw adductor muscle on the mandible. An equivalent cartilage is absent in chick and quail. We hypothesize that species-specific jaw architecture and mechanical forces promote secondary cartilage in duck through the differential regulation of FGF and TGF{beta} signaling. First, we perform transplants between chick and duck embryos and demonstrate that the ability of neural crest mesenchyme (NCM) to direct the species-specific insertion of muscle and the formation of secondary cartilage depends upon the amount and spatial distribution of NCM-derived connective tissues. Second, we quantify motility and build finite element models of the jaw complex in duck and quail, which reveals a link between species-specific jaw architecture and the predicted mechanical force environment. Third, we investigate the extent to which mechanical load mediates FGF and TGF{beta} signaling in the duck jaw adductor insertion, and discover that both pathways are mechano-responsive and required for secondary cartilage formation. Additionally, we find that FGF and TGF{beta} signaling can also induce secondary cartilage in the absence of mechanical force or in the adductor insertion of quail embryos. Thus, our results provide novel insights on molecular, cellular, and biomechanical mechanisms that couple musculoskeletal form and function during development and evolution.

developmental biology↗

bioRxiv2017

Scalable and efficient single-cell DNA methylation sequencing by combinatorial indexing.

Here we present a novel method: single-cell combinatorial indexing for methylation analysis (sci-MET), which is the first highly scalable assay for whole genome methylation profiling of single cells. We use sci-MET to produce 2,697 total single-cell bisulfite sequencing libraries and achieve read alignment rates of 69 {+/-} 7%, comparable to those of bulk cell methods. As a proof of concept, we applied sci-MET to successfully deconvolve the cellular identity of a mixture of three human cell lines.

genomics↗