Biology subjects

Yang, Y. A.

Publications and source records attributed to Yang, Y. A..

4 recordsLinked to original sources

bioRxiv2025

Simultaneous single-cell CRISPR, RNA, and ATAC-seq enables multiomic CRISPR screens to identify gene regulatory relationships

The ability to identify gene functions and interactions in specific cellular contexts has been greatly enabled by functional genomics technologies. CRISPR-based genetic screens have proven invaluable in elucidating gene function in mammalian cells. Single-cell functional genomics methods, such as Perturb-seq and Spear-ATAC, have made it possible to achieve high-throughput mapping of the functional effects of gene perturbations by profiling transcriptomes and DNA accessibility, respectively. Combining single-cell chromatin accessibility and transcriptomic data via multiomic approaches has facilitated the discovery of novel cis and gene regulatory interactions. However, pseudobulk readouts from cell populations can often cloud the interpretation of results due to a heterogeneous response from cells receiving the same genetic perturbation, which could be mitigated by using transcriptional profiles of single cells to subset the ATAC-seq data. Existing methods to capture CRISPR guide RNAs to simultaneously assess the impact of genetic perturbations on RNA and ATAC profiles require either cloning of gRNA libraries in specialized vectors or implementing complex protocols with multiple rounds of barcoding. Here, we introduce CAT-ATAC, a technique that adds CRISPR gRNA capture to the existing 10X Genomics Multiome assay, generating paired transcriptome, chromatin accessibility and perturbation identity data from the same individual cells. We demonstrate up to 77% guide capture efficiency for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we were able to identify a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss of function experiments, we further validated that the gene, ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal novel gene and cellular interactions and functions.

genomics↗

bioRxiv2025

SAMHD1 Knockout iPSC model enables high lenti-viral transduction in myeloid cell types

Recent advances in functional genomics tools have ushered in a new era of genetic editing to identify molecular pathways relevant to developmental and disease biology. However, limited model systems are available that adequately mimic cell states and phenotypes associated with human disease pathways. Here, we quantitatively analyzed the founder population bottleneck effect and demonstrated how the population changes from induced pluripotent stem cells (iPSCs) to hematopoietic stem cells and to the final induced macrophage population. We then engineered SAMHD1 knockout (KO) iPSC and characterized the iPSC line with RNA Seq, and induced macrophages from two distinct protocols with functional analysis. We then generated SAMHD1 KO CRISPR-dCAS9 KRAB iPSC through lenti-viral transduction aiming to increase the efficiency of lentiviral mediated gene transfer. We demonstrated increased lenti-viral transduction efficiency in induced macrophage, as well as microglia induced with two distinct protocols. This model allows for efficient gene knock down, as well as large-scale functional genomics screens in mature iPSC-derived macrophages or microglia with applications in innate immunity and chronic inflammatory disease biology. These experiments highlight the broad applicability of this platform for disease-relevant target identification and may improve our ability to run large-scale screens in iPSC-derived myeloid model systems.

cell biology↗

bioRxiv2024

Notch signaling maintains a progenitor-like subclass of hepatocellular carcinoma

Hepatocellular carcinomas (HCCs) constitute one of the few cancer indications for which mortality rates continue to rise. While Notch signaling dictates a key progenitor lineage choice during development, its role in HCC has remained controversial. Using therapeutic antibodies targeting Notch ligands and receptors to screen over 40 patient-derived xenograft models, we here identify progenitor-like HCCs that crucially depend on a tumor-intrinsic JAG1-NOTCH2 signal. Inhibiting this signal induces tumor regressions by triggering progenitor-to-hepatocyte differentiation, the same cell fate-switch that Notch controls during development. Transcriptomic analysis places the responsive tumors within the well-characterized progenitor subclass, a poor prognostic group of highly proliferative tumors, providing a diagnostic method to enrich for Notch-dependent HCCs. Furthermore, single-cell RNA sequencing uncovers a heterogeneous population of tumor cells and reveals how Notch inhibition shifts cells from a mixed cholangiocyte-hepatocyte lineage to one resembling mature hepatocytes. Analyzing the underlying transcriptional programs brings molecular detail to this process by showing that Notch inhibition de-represses expression of CEBPA, which enables the activity of HNF4, a hepatocyte lineage factor that is otherwise quiescent. We thus describe a compelling and targetable dependency in a poor-prognosis class of HCCs.

cancer biology↗

bioRxiv2024

Dermatopontin-expressing fibroblasts mediate an essential skin macrophage niche

Fibroblasts are present in all tissues and are crucial for maintaining tissue homeostasis. We previously identified fibroblasts marked by Dermatopontin (Dpt) but their role in supporting macrophage homeostasis remains unclear. Here, we generated novel mesenchymal lineage-restricted genetic tools to target Dpt expressing fibroblasts and elucidate their role in supporting skin macrophages. Transcriptional profiling, flow cytometry, and in situ hybridization uncovered two broad populations of F4/80-expressing skin macrophages, denoted by high expression of CD206 and CD64 (CD206hiCD64+), or CD11c. Targeted depletion of Dpt+ fibroblasts resulted in a profound loss of both macrophage populations. Conditional deletion of colony-stimulating factor-1 (Csf1) in Dpt+ fibroblasts revealed that CD206hiCD64+, and not CD11c+, macrophages are acutely dependent on fibroblast-derived Csf1, consistent with their higher expression of the Csf1 receptor. Following Csf1 deletion in Dpt+ fibroblasts, loss of CD206hiCD64+ macrophages were observed across the dermis, dermal white adipose tissue (dWAT), and adventitia, accompanied by a modest upregulation of fibroblast-related and extracellular matrix (ECM) genes and structural changes to the skin. Alterations to the skin network upon loss of fibroblast-derived Csf1 and CD206hiCD64+ macrophages led to a significant delay in wound healing. We also demonstrate the CSF1-CSF1R signaling pathway is functionally relevant in human systemic sclerosis, or scleroderma, as elevated levels of CSF1 produced by fibroblasts and an increased abundance of macrophages both correlate with disease severity. Our findings demonstrate the role of Dpt+ fibroblasts in regulating a Csf1-dependent macrophage niche in skin and orchestrating responses in injury and disease.

immunology↗