Biology subjects

Chou, C.-L.

Publications and source records attributed to Chou, C.-L..

11 recordsLinked to original sources

bioRxiv2026

Kinome-wide CRISPR/Cas9-knockout screening reveals critical protein kinases in vasopressin V2-receptor signaling

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). Positive regulators (n=14) included PKA-catalytic subunit (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type I, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-{beta} receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. TGF-{beta} exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-{beta}-mediated vasopressin escape. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Significance StatementCells throughout the body are regulated by extracellular signals like the hormone, vasopressin. Hormonal effects on cellular function are mediated by membrane receptors that trigger biochemical changes, often by inducing chemical modification of the amino acids making up individual proteins, such as addition of function-altering phosphate groups (phosphorylation). Protein phosphorylation is mediated by enzymes known as "protein kinases". Here, we have screened all known protein kinases using modern CRISPR/Cas9 technology to identify those involved in vasopressin action in the kidney. As expected from prior knowledge, the screen identified protein kinase A and one of its regulatory subunits, but also identified several protein kinases not previously implicated in vasopressin action in the kidney.

systems biology↗

bioRxiv2025

Single-cell RNA-seq using UltraMarathonRT expands theknown transcriptome

The ability to map messenger RNA (mRNA) molecules from individual cells using next-generation sequencing technologies, known as single-cell RNA-seq (scRNA-seq), is transforming biology by redefining cellular identities with unmatched detail. However, all current protocols depend on copying RNA into complementary DNA with a single reverse transcriptase (RT) derived from murine leukemia virus, which is an RT enzyme known for low processivity and limited ability to unfold complex RNA structures. Here, for the first time, we introduce a group II intron reverse transcriptase, UltraMarathonRT (uMRT), to perform scRNA-seq. We demonstrate that this enzyme reveals an unexpected transcriptomics landscape by capturing additional genes and other genomic features that conventional RTs miss. We also combined uMRT with metabolic RNA labeling, nucleoside conversion and scRNA-seq to explore genome-wide transcriptome dynamics at the single-cell level. Overall, we establish uMRT as a transformative biotechnological tool for single-cell transcriptomics.

molecular biology↗

bioRxiv2025

Vasopressin-Dependent β-Catenin Phosphorylation at Ser552 and Branching Structure of Mouse Collecting Duct System

BackgroundPhosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases PKA-dependent phosphorylation of {beta}-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. MethodsTo address the role of {beta}-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. ResultsThe mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 (AQP2) or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 {+/-} 0.66 in control mice to 3.33 {+/-} 0.82 in Ser552Ala mutant mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between control and Ser552Ala mutant mice (both [~]13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two CDK inhibitor proteins, Cdkn1b and Cdkn1c. At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. ConclusionsThe observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of {beta}-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla. Key pointsO_LIVasopressin regulates collecting duct (CD) transport by triggering phosphorylation of multiple proteins including {beta}-catenin at Ser552. C_LIO_LIMutating Ser552 to a non-phosphorylable amino acid in mice resulted in altered CD branching without loss of differentiation in adult CDs. C_LIO_LIThe findings point to a role for {beta}-catenin Ser552 phosphorylation in CD branching and sub-segmental CD elongation. C_LI

physiology↗

bioRxiv2025

CREB-family transcription factors and vasopressin-mediated regulation of Aqp2 gene transcription

BackgroundWater homeostasis is regulated by the peptide hormone arginine vasopressin (AVP), which promotes water reabsorption in the renal collecting duct. The regulation of Aqp2 gene transcription is a key mechanism through which AVP modulates water transport as disruption of this mechanism leads to water balance disorders. Therefore, an important goal is to understand the regulatory processes that control Aqp2 gene transcription. While CREB (CREB1) has been proposed as the primary transcription factor responsible for Aqp2 transcription, recent evidence challenges this view, suggesting that other CREB-like transcription factors, including ATF1 and CREM, may play a role. MethodsWe employed the CRISPR/Cas9 gene-editing system to delete Atf1, Creb1, and Crem in mpkCCD cells, an immortalized mouse collecting duct cell line. These cell lines were then exposed to the vasopressin analog, dDAVP, to assess the role of these transcription factors in regulating Aqp2 expression. AQP2 protein levels were measured by immunoblotting and RNA-seq was used to analyze changes in Aqp2 mRNA abundance, as well as other transcriptomic changes. ResultsDeletion of all three transcription factors (ATF1, CREB1, and CREM) led to a significant reduction in the vasopressin-induced upregulation of AQP2 protein, confirming their role in regulating Aqp2 expression. RNA-seq data showed that Aqp2 mRNA levels mirrored changes in protein abundance, supporting the idea that these transcription factors affect Aqp2 transcription. Rescue experiments in triple knockout cells showed that expressing any of the three transcription factors restored the response to vasopressin. ConclusionsOur findings demonstrate that ATF1, CREB1, and CREM have redundant roles in regulating Aqp2 transcription. Based on these results and prior data, we propose that these CREB-family transcription factors may regulate Aqp2 gene transcription indirectly by controlling the expression of additional unidentified transcription factors. Key PointsO_LICREB-family transcription factors (ATF1, CREB1, and CREM) were deleted in mpkCCD cells to assess their roles in Aqp2 gene transcription C_LIO_LICRISPR/Cas9 knockout of all three transcription factors strongly reduced the ability of vasopressin to increase AQP2 mRNA and protein C_LIO_LIRe-expression of any of the three restored the vasopressin response indicating redundant roles of the three transcription factors C_LI

physiology↗

bioRxiv2025

Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis

BackgroundAtherosclerosis occurs preferentially in the arteries exposed to disturbed flow (d-flow), while the stable flow (s-flow) regions are protected even under hypercholesterolemic conditions. We recently showed that d-flow alone initiates flow-induced reprogramming of endothelial cells (FIRE), including the novel concept of partial endothelial-to-immune-cell-like transition (partial EndIT), but was not validated using a genetic lineage-tracing model. Here, we tested and validated the two-hit hypothesis that d-flow is an initial instigator of partial FIRE but requires hypercholesterolemia to induce a full-blown FIRE and atherosclerotic plaque development. MethodsMice were treated with adeno-associated virus expressing proprotein convertase subtilisin/kexin type 9 and a Western diet to induce hypercholesterolemia and/or partial carotid ligation (PCL) surgery to expose the left common carotid artery (LCA) to d-flow. Single-cell RNA sequencing (scRNA-seq) analysis was performed using cells obtained from the intima and leftover LCAs and the control right common carotid arteries at 2 and 4 weeks post-PCL. Comprehensive immunohistochemical staining was performed on EC-specific confetti mice treated with PCL and hypercholesterolemic conditions at 4 weeks post-PCL to validate endothelial reprogramming. ResultsAtherosclerotic plaques developed by d-flow under hypercholesterolemia at 2 and 4 weeks post-PCL, but not by d-flow or hypercholesterolemia alone, as expected. The scRNA-seq results of 98,553 single cells from 95 mice revealed 25 cell clusters; 5 EC, 3 vascular smooth muscle cell (SMC), 5 macrophage (M{Phi}), and additional fibroblast, T cell, natural killer cell, dendritic cell, neutrophil, and B cell clusters. Our scRNA-seq analyses showed that d-flow under hypercholesterolemia transitioned healthy ECs to full immune-like (EndIT) and, more surprisingly, foam cells (EndFT), in addition to inflammatory and mesenchymal cells (EndMT). Further, EC-derived foam cells shared remarkably similar transcriptomic profiles with foam cells derived from SMCs and M{Phi}s. Comprehensive lineage-tracing studies using immunohistochemical staining of canonical protein and lipid markers in the EC-specific confetti mice clearly demonstrated direct evidence supporting the novel FIRE hypothesis, including EndIT and EndFT, when d-flow was combined with hypercholesterolemia. Further, reanalysis of the publicly available human carotid plaque scRNA-seq and Perturb-seq datasets supported the FIRE hypothesis and a potential mechanistic link between the genes and FIRE. ConclusionWe provide evidence supporting the two-hit hypothesis: ECs in d-flow regions, such as the branching points, are partially reprogrammed, while hypercholesterolemia alone has minimal endothelial reprogramming effects. Under hypercholesterolemia, d-flow fully reprograms arterial ECs, including the novel EndIT and EndFT, in addition to inflammation and EndMT, during atherogenesis. This single-cell atlas provides a crucial roadmap for developing novel mechanistic understanding and therapeutics targeting flow-sensitive genes, proteins, and pathways of atherosclerosis.

cell biology↗

bioRxiv2022

Early Signaling Events in Renal Compensatory Hypertrophy Revealed by Multi-Omics

Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we used a multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule. Morphometry applied to microdissected proximal tubules showed that growth of the proximal tubule involves a marked, rapid increase in cell volume rather than cell number. Measurements of DNA accessibility (ATAC-seq), transcriptome (RNA-seq) and proteome (quantitative protein mass spectrometry) independently identified patterns of change that are indicative of activation of the lipid-regulated transcription factor, PPAR. Activation of PPAR by fenofibrate administration increased proximal tubule cell size, while genetic deletion of PPAR in mice decreased it. The results indicate that PPAR is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.

systems biology↗

bioRxiv2022

Circadian Gene Expression in Mouse Renal Proximal Tubule

Circadian variability in kidney function has long been recognized but is often ignored as a potential confounding variable in in vivo physiological experiments. To provide a guide for physiological studies on the kidney proximal tubule, we have now created a data resource consisting of expression levels for all measurable mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. This approach employs small-sample RNA-sequencing (RNA-seq) applied to microdissected renal proximal tubules including both S1 proximal convoluted tubules (PCTs) and S2 proximal straight tubules (PSTs). The data were analyzed using JTK-Cycle to detect periodicity. The data are provided as a user-friendly web page at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox/. In PCTs, 234 transcripts were found to vary in a circadian manner (3.7 % of total quantified). In PSTs, 334 transcripts were found to vary in a circadian manner (5.3 % of total quantified). Transcripts previously known to be associated with corticosteroid action and transcripts associated with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day (Zeitgeber 14-22), corresponding to the peak levels of corticosterone and glomerular filtration rate in mice. BlurbCircadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-seq in microdissected proximal S1 and S2 segments to identify transcripts that vary in a circadian manner. The data were used to construct a user-friendly web resource.

systems biology↗

bioRxiv2020

Phosphoproteomic Identification of Vasopressin-Regulated Protein Kinases in Collecting Duct Cells

Background and PurposeThe peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a protein kinase A (PKA)-dependent signaling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. Experimental ApproachWe carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct cells (mpkCCD). Quantification was done using stable isotope labeling (SILAC method). Key Results9640 phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups distinguished predominantly by the amino acids at positions +1, -3, -2 and -5 relative to the phosphorylated amino acid. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six of them plus PKA appeared to account for phosphorylation of more than 80% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (PCTAIRE-3, Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (ERK1; Mapk3), and myosin light chain kinase (Mylk). Conclusion and ImplicationsIn V2 receptor-mediated signaling, PKA is at the head of a complex network that includes at least 6 downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data generated in this study is provided as a web-based data resource for future studies of G-protein coupled receptors.

systems biology↗

bioRxiv2020

Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance

A major objective in modern biology is generation of comprehensive atlases of various organs identifying all cell types and their expressed genes. In kidney, extensive data exists for proximal tubule and collecting duct cells, but not for non-abundant intermediate epithelial cell types. Here, we coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa. Clusters containing Slc12a3+/Pvalb+ and Slc12a3+/Pvalb- cells were identified as DCT1 and DCT2 cells. The DCT1 cells appear to be heterogeneously associated with variable expression of Slc8a1, Calb1, and Ckb among other mRNAs. No DCT2-specific transcripts were found. The analysis also identified two distinct cell types in the Slc12a1+ portion of Henles loop as well as Nos1+/Avpr1a+ macula densa cells. Thus, we identify unexpected cell diversity in the intermediate region of the nephron and create a web-based data resource for these cells.

systems biology↗

bioRxiv2018

Cell-Type Selective Markers Represented in Whole-Kidney RNA-Seq Data

Bulk-tissue RNA-Seq is seeing increasing use in the study of physiological and pathophysiological processes in the kidney. However, the presence of multiple cell types in kidney complicates the data interpretation. Here we address the question, \"What cell types are represented in whole-kidney RNA-Seq data?\" to identify circumstances in which bulk-kidney RNA-Seq can be successfully interpreted. We carried out RNA-Seq in mouse whole kidneys and microdissected renal tubule segments. To aid in the interpretation of the data, we compiled a database of cell-type selective protein markers for 43 cell types believed to be present in kidney tissue. The whole-kidney RNA-Seq analysis identified transcripts corresponding to 17742 genes, distributed over 5 orders of magnitude of expression level. Markers for all 43 curated cell types were detectable. Analysis of the cellular makeup of mouse and rat kidney, calculated from published literature, suggests that proximal tubule cells account for more than half of the mRNA in a kidney. Comparison of RNA-Seq data from microdissected proximal tubules with whole-kidney data supports this view. RNA-Seq data for cell-type selective markers in bulk-kidney samples provide a valid means to identify changes in minority-cell abundances in kidney tissue. Because proximal tubules make up a substantial fraction of whole-kidney samples, changes in proximal tubule gene expression can be assessed presumptively by bulk-kidney RNA-Seq, although results could potentially be obscured by the presence of mRNA from other cell types. The dominance of proximal tubule cells in whole-kidney samples also has implications for the interpretation of single-cell RNA-Seq data.

systems biology↗

bioRxiv2017

Transcriptomes of major renal collecting-duct cell types in mouse identified by single-cell RNA-Seq

Prior RNA sequencing (RNA-Seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume and extracellular fluid composition. To enrich these cell types, we used fluorescence-activated cell sorting (FACS) that employed well established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, viz. c-Kit. Single-cell RNA-Seq using the 1C- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs and PCs. The data were used to create a freely-accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type including cell-surface receptors, transcription factors, transporters and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing both aquapor{inverted exclamation}n-2 and either anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g. Notch2 chiefly in PCs vs Jag1 chiefly in ICs) suggesting signaling crosstalk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.\n\nSIGNIFICANCE STATEMENTA long-term goal in mammalian biology is to identify the genes expressed in every cell type of the body. In kidney, the expressed genes (\"transcriptome\") of all epithelial cell types have already been identified with the exception of the cells that make up the renal collecting duct, responsible for regulation of blood pressure and body fluid composition. Here, a technique called \"single-cell RNA-Seq\" was used in mouse to identify transcriptomes for the major collecting-duct cell types: type A intercalated cells, type B intercalated cells and principal cells. The information was used to create a publicly-accessible online resource. The data allowed identification of genes that are selectively expressed in each cell type, informative for cell-level understanding of physiology and pathophysiology.

physiology↗