Biology subjects

Civelek, M.

Publications and source records attributed to Civelek, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Sex-Specific Regulation of Endothelial-to-Mesenchymal Transition (EndMT) During Atherosclerosis

Sex differences in atherosclerotic plaque biology underlie clinically distinct manifestations of acute coronary syndromes, yet the molecular mechanisms driving these differences remain incompletely understood. Endothelial-to-mesenchymal transition (EndMT) is increasingly recognized as a contributor to plaque remodeling, but whether EndMT is regulated in a sex-specific and stage-dependent manner across atherosclerosis has not been systematically examined. Here, we integrated bulk RNA sequencing with single-cell transcriptomic analyses in endothelial cells and human atherosclerotic plaques to define sex-specific EndMT regulation across healthy and advanced-stage disease. Using trajectory analyses in endothelial cells undergoing EndMT, we observed pronounced sex-specific regulation in healthy endothelium, with females exhibiting stronger early EndMT activation, whereas endothelial cells derived from atherosclerotic plaques displayed markedly attenuated sex differences with disease progression. Consistent sex-divergent pseudotime trajectories were observed in human carotid plaque endothelial single-cell RNA-seq data, with females showing greater EndMT activation at earlier stages and males at later stages. Together, these findings support a stage-dependent model of sex-specific EndMT regulation, indicating that the functional consequences of EndMT are highly context dependent and may differ across early and late disease stages. Integration of these datasets prioritized high-confidence sex-specific EndMT regulators, including COL4A1, PECAM1, CD151, JAG1, FN1, NEDD9, PODXL, MAFB, PROCR, and CDH13, providing a mechanistic framework to explain clinically observed sex differences in plaque biology and to guide targeted functional interrogation.

genomics↗

bioRxiv2025

Female-biased vascular smooth muscle cell gene regulatory networks predict MYH9 as a key regulator of fibrous plaque phenotype

Atherosclerosis, a chronic inflammatory condition driving coronary artery disease (CAD), manifests in two primary plaque types: unstable atheromatous plaques and stable fibrous plaques. While significant research has focused on atheromatous plaques, recent studies emphasize the growing importance of fibrous plaques, particularly in females under 50 years of age, where erosion on fibrous plaques significantly contributes to coronary thrombosis. The molecular mechanisms underlying sex differences in atherosclerotic plaque characteristics, including vascular smooth muscle cell (VSMC) contributions, remain understudied. Therefore, we utilized sex-specific gene regulatory networks (GRNs) derived from VSMC gene expression data from 119 male and 32 female heart transplant donors to identify molecular drivers of fibrous plaques. GRN analysis revealed two female-biased networks in VSMC, GRNfloralwhite and GRNyellowgreen, enriched for inflammatory signaling and actin remodeling pathways, respectively. Single-cell RNA sequencing of carotid plaques from female and male patients confirmed the sex specificity of these networks in VSMCs. Further sub cellular phenotyping of the single-cell RNA sequencing revealed a sex-specific gene expression signature within GRNyellowgreen for VSMCs enriched for contractile and vasculature development pathways. Bayesian network modeling of the GRNyellowgreen identified MYH9 as a key driver gene. Indeed, elevated MYH9 protein expression in atherosclerotic plaques was associated with higher smooth muscle cell content and lower lipid content in female plaques, suggesting its involvement in fibrous plaque formation. Further proteomic analysis confirmed MYH9s upregulation in female fibrous plaques only and its correlation with stable plaque features. These findings provide novel insights into sex-specific molecular mechanisms regulating fibrous plaque formation.

genomics↗

bioRxiv2024

Fibroblast mechanoperception instructs pulmonary developmental and pattern specification gene expression programs

Dysregulation of the cellular mechanisms that coordinate the interpretation and transduction of microenvironmental biophysical signals are a unifying feature of tissue remodeling pathologies such as fibrosis and cancer. While genomic regulation downstream of normal mechanotransduction (i.e. cases where cells sense soft and stiff appropriately) is well studied, significantly less is known about the consequences of abnormal mechanoperception and subsequent misinterpretation of the mechanical environment. Leveraging Thy-1 (a.k.a. CD90) loss as a model of impaired mechanoperception, we employed ATAC- and RNA-sequencing in parallel to characterize the changes in lung fibroblast genomic activity in response to a combination of substrate stiffness and culture time. Notably, we find perturbed mechanoperception elicits a near-complete shutdown of HOXA5, a transcription factor responsible for pattern specification and development in the nascent lung. In vitro investigation of HOXA5 expression reveals a potential mechanism connecting increased v integrin signaling, cytoskeletal tension, and SRC kinase activity to HOXA5 silencing. These results establish novel links between integrin signaling and the expression dynamics of genes necessary for tissue formation and regeneration in the injured and/or developing lung, particularly HOXA5.

cell biology↗

bioRxiv2024

Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression

Coronary artery disease (CAD) is a complex disorder with genetic and environmental influences. Genome-wide association studies (GWAS) have identified over 300 genomic loci associated with disease risk. However, identifying the functional variants within these loci has been limited in large part due to linkage disequilibrium. This represents a critical step in understanding the molecular mechanisms underlying disease risk. To identify and prioritize candidate causal CAD-associated variants, we performed lentivirus-based massively parallel reporter assays (lentiMPRAs) in primary vascular smooth muscle cells (SMCs), which play significant roles in atherosclerosis, the underlying cause of CAD. We tested 25892 CAD-associated variants for their allele-specific enhancer activity in quiescent and proliferative SMCs, modeling healthy and disease conditions. We identified 122 candidate variants showing significant enhancer activity and differences in reporter gene expression between risk and non-risk alleles. We also identified 23 variants showing condition-biased allelic imbalance and 41 variants showing sex-biased allelic imbalance. We further functionally characterized 25892 variants by performing CUT&RUN assays to identify variants in enhancer and promoter regions of SMCs. By integrating the results of these experiments, we identified a credible set of 49 CAD-associated variants in functionally relevant regions. Furthermore, by comparing these identified variants with our previously obtained expression quantitative trait loci (eQTL) data, 27 of these 49 variants were associated with SMC gene expression levels. Finally, we performed CRISPRi experiments on 8 variants comprising 9 variant-gene pairs, rs35976034 (MAP1S), rs4888409 (CFDP1), rs73193808 (MAP3K7CL), rs67631072 (INPP5B/FHL3), rs1651285 (SNHG18), rs17293632 (SMAD3), rs2238792 (ARVCF), rs4627080 (NRIP3), to confirm their regulatory potential of nearby gene expression. Taken together, our results comprehensively fine-map the causal variants that confer increased risk of CAD through their effects on vascular smooth muscle cells.

genomics↗

bioRxiv2024

14-3-3ζ regulates adipogenesis by modulating chromatin accessibility during the early stages of adipocyte differentiation

We previously established the scaffold protein 14-3-3{zeta} as a critical regulator of adipogenesis and adiposity, but the temporal specificity of its action during adipocyte differentiation remains unclear. To decipher if 14-3-3{zeta} exerts its regulatory functions on mature adipocytes or on adipose precursor cells (APCs), we generated Adipoq14-3-3{zeta}KO and Pdgfra14-3-3{zeta}KO mouse models. Our findings revealed a pivotal role for 14-3-3{zeta} in APC differentiation in a sex-dependent manner, whereby male and female Pdgfra14-3-3{zeta}KO mice display impaired or potentiated weight gain, respectively, as well as fat mass. To better understand how 14-3-3{zeta} regulates the adipogenic transcriptional program in APCs, CRISPR-Cas9 was used to generate TAP-tagged 14-3-3{zeta}-expressing 3T3-L1 preadipocytes. Using these cells, we examined if the 14-3-3{zeta} nuclear interactome is enriched with adipogenic regulators during differentiation. Regulators of chromatin remodeling, such as DNMT1 and HDAC1, were enriched in the nuclear interactome of 14-3-3{zeta}, and their activities were impacted upon 14-3-3{zeta} depletion. The interactions between 14-3-3{zeta} and chromatin-modifying enzymes suggested that 14-3-3{zeta} may control chromatin remodeling during adipogenesis, and this was confirmed by ATAC-seq, which revealed that 14-3-3{zeta} depletion impacted the accessibility of up to 1,244 chromatin regions corresponding in part to adipogenic genes, promoters, and enhancers during the initial stages of adipogenesis. Moreover, 14-3-3{zeta}-dependent chromatin accessibility was found to directly correlate with the expression of key adipogenic genes. Altogether, our study establishes 14-3-3{zeta} as a crucial epigenetic regulator of adipogenesis and highlights the usefulness of deciphering the nuclear 14-3-3{zeta} interactome to identify novel pro-adipogenic factors and pathways.

physiology↗

bioRxiv2023

Systems genetics analysis of human body fat distribution genes identifies Wnt signaling and mitochondrial activity in adipocytes

BACKGROUNDExcess fat in the abdomen is a sexually dimorphic risk factor for cardio-metabolic disease. The relative storage between abdominal and lower-body subcutaneous adipose tissue depots is approximated by the waist-to-hip ratio adjusted for body mass index (WHRadjBMI). Genome-wide association studies (GWAS) identified 346 loci near 495 genes associated with WHRadjBMI. Most of these genes have unknown roles in fat distribution, but many are expressed and putatively act in adipose tissue. We aimed to identify novel sex- and depot-specific drivers of WHRadjBMI using a systems genetics approach. METHODSWe used two independent cohorts of adipose tissue gene expression with 362 - 444 males and 147 - 219 females, primarily of European ancestry. We constructed sex- and depot- specific Bayesian networks to model the gene-gene interactions from 8,492 adipose tissue genes. Key driver analysis identified genes that, in silico and putatively in vitro, regulate many others, including the 495 WHRadjBMI GWAS genes. Key driver gene function was determined by perturbing their expression in human subcutaneous pre-adipocytes using lenti-virus or siRNA. RESULTS51 - 119 key drivers in each network were replicated in both cohorts. We used single-cell expression data to select replicated key drivers expressed in adipocyte precursors and mature adipocytes, prioritized genes which have not been previously studied in adipose tissue, and used public human and mouse data to nominate 53 novel key driver genes (10 - 21 from each network) that may regulate fat distribution by altering adipocyte function. In other cell types, 23 of these genes are found in crucial adipocyte pathways: Wnt signaling or mitochondrial function. We selected seven genes whose expression is highly correlated with WHRadjBMI to further study their effects on adipogenesis/Wnt signaling (ANAPC2, PSME3, RSPO1, TYRO3) or mitochondrial function (C1QTNF3, MIGA1, PSME3, UBR1). Adipogenesis was inhibited in cells overexpressing ANAPC2 and RSPO1 compared to controls. RSPO1 results are consistent with a positive correlation between gene expression in the subcutaneous depot and WHRadjBMI, therefore lower relative storage in the subcutaneous depot. RSPO1 inhibited adipogenesis by increasing {beta}-catenin activation and Wnt-related transcription, thus repressing PPARG and CEBPA. PSME3 overexpression led to more adipogenesis than controls. In differentiated adipocytes, MIGA1 and UBR1 downregulation led to mitochondrial dysfunction, with lower oxygen consumption than controls; MIGA1 knockdown also lowered UCP1 expression. SUMMARYANAPC2, MIGA1, PSME3, RSPO1, and UBR1 affect adipocyte function and may drive body fat distribution.

systems biology↗

bioRxiv2023

Female gene networks are expressed in myofibroblast-like smooth muscle cells in vulnerable atherosclerotic plaques.

Women presenting with coronary artery disease (CAD) more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. Here, we show sex-stratified gene regulatory networks (GRNs) from human carotid atherosclerotic tissue. Prioritization of these networks identified two main SMC GRNs in late-stage atherosclerosis. Single-cell RNA-sequencing mapped these GRNs to two SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like GRN was mostly expressed in plaques that were vulnerable in females. Finally, mice orthologs of the female myofibroblast-like genes showed retained expression in advanced plaques from female mice but were downregulated in male mice during atherosclerosis progression. Female atherosclerosis is driven by GRNs that promote a fibrous vulnerable plaque rich in myofibroblast-like SMCs.

bioinformatics↗

bioRxiv2022

FHL5 controls vascular disease-associated gene programs in smooth muscle cells

BackgroundGenome-wide association studies (GWAS) have identified hundreds of loci associated with common vascular diseases such as coronary artery disease (CAD), myocardial infarction (MI), and hypertension. However, the lack of mechanistic insights for a majority of these loci limits translation of these findings into the clinic. Among these loci with unknown functions is UFL1-FHL5 (chr6q16.1), a locus that reached genome-wide significance in a recent CAD/MI GWAS meta-analysis. In addition to CAD/MI, UFL1-FHL5 is also implicated to coronary calcium, intracranial aneurysm, and migraine risk, consistent with the widespread pleiotropy observed among other GWAS loci. MethodsWe apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and imaging of human coronary artery tissues to implicate Four-and-a-half LIM domain 5 (FHL5) as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments. ResultsWe prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through eQTL colocalization methods. FHL5 gene expression was enriched in the SMC and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating SMC contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream CAD/MI loci, such as FOXL1 and FN1 that have roles in vascular remodeling. ConclusionTaken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling loci. These trans-acting mechanisms may account for a portion of the heritable risk for complex vascular diseases.

genomics↗

bioRxiv2022

Network preservation analysis reveals dysregulated metabolic pathways in human vascular smooth muscle cell phenotypic switching

Vascular smooth muscle cells (VSMCs) are key players involved in atherosclerosis, the underlying cause of coronary artery disease (CAD). They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. An in-depth characterization of their gene regulatory networks can help better understand how their dysfunction may impact disease progression. We conducted a gene expression network preservation analysis in aortic SMCs isolated from 151 multi-ethnic heart transplant donors cultured under quiescent or proliferative conditions. We identified 86 groups of co-expressed genes (modules) across the two conditions and focused on the 18 modules that are least preserved between the phenotypic conditions. Three of these modules were significantly enriched for genes belonging to proliferation, migration, cell adhesion, and cell differentiation pathways, characteristic of phenotypically modulated proliferative VSMCs. The majority of the modules, however, were enriched for metabolic pathways consisting of both nitrogen-related and glycolysis-related processes. Therefore, we explored correlations between nitrogen metabolism-related genes and CAD-associated genes and found significant correlations, suggesting the involvement of the nitrogen metabolism pathway in CAD pathogenesis. for six genes in the nitrogen metabolism pathway. We also created gene regulatory networks enriched for genes in glycolysis and predicted key regulatory genes driving glycolysis dysregulation. Our work suggests that dysregulation of VSMC metabolism participates in phenotypic transitioning, which may contribute to disease progression and suggests that AMT and MPI may play an important role in regulating nitrogen and glycolysis related metabolism in SMCs.

systems biology↗

bioRxiv2022

Predicting mechanisms of action at genetic loci associated with discordant effects on type 2 diabetes and abdominal fat accumulation

Metabolic syndrome (MetSyn) is a cluster of dysregulated metabolic conditions that occur together to increase the risk for cardiometabolic disorders such as type 2 diabetes (T2D). One key condition associated with MetSyn, abdominal obesity, is measured by computing the ratio of waist-to-hip circumference adjusted for the body-mass index (WHRadjBMI). WHRadjBMI and T2D are complex traits with genetic and environmental components, which has enabled genome-wide association studies (GWAS) to identify hundreds of loci associated with both. Statistical genetics analyses of these GWAS have predicted that WHRadjBMI is a strong causal risk factor of T2D and that these traits share genetic architecture at many loci. To date, no variants have been described that are simultaneously associated with protection from T2D but with increased abdominal obesity. Here, we used colocalization analysis to identify genetic variants with a shared association for T2D and abdominal obesity. This analysis revealed the presence of five loci associated with discordant effects on T2D and abdominal obesity. The alleles of the lead genetic variants in these loci that were protective against T2D were also associated with increased abdominal obesity. We further used publicly available expression, epigenomic, and genetic regulatory data to predict the effector genes (eGenes) and functional tissues at the 2p21, 5q21.1, and 19q13.11 loci. We also computed the correlation between the subcutaneous adipose tissue (SAT) expression of predicted effector genes (eGenes) with metabolic phenotypes and adipogenesis. We proposed a model to resolve the discordant effects at the 5q21.1 locus. We find that eGenes gypsy retrotransposon integrase 1 (GIN1), diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2), and peptidylglycine alpha-amidating monooxygenase (PAM) represent the likely causal eGenes at the 5q21.1 locus. Taken together, these results are the first to describe a potential mechanism through which a genetic variant can confer increased abdominal obesity but protection from T2D risk. Understanding precisely how and which genetic variants confer increased risk for MetSyn will develop the basic science needed to design novel therapeutics for metabolic syndrome.

genomics↗

bioRxiv2022

Genetic Regulation of Circular RNA Expression in Human Aortic Smooth Muscle Cells and Vascular Traits

BackgroundCircular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease by differentiating from a quiescent to proliferative phenotype. The role of circRNAs in SMCs and their relevance to cardiovascular disease is largely unexplored. ResultsIn this study, we employ a systems genetics approach to identify circRNA transcripts at a genome wide level and their relevance in cardiovascular traits. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from multiethnic donors. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 circRNAs which were differentially expressed. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs which were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not in quiescent SMCs. We identified 6 SNPs which had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease related GWAS loci. We identified 7 blood pressure, 1 myocardial infarction, and 3 coronary artery disease loci which were associated with a circRNA transcript (circZKSCAN1, circFOXK2, circANKRD36, circLARP4, circCEP85L, circGTF3C2, circPDS5A, circSLC4A7, and chr17:42610108|42659552) but not mRNA transcript. ConclusionsOverall, our results provide mechanistic insight into the regulation of circRNA expression and the genetic basis of cardiovascular disease.

genetics↗

bioRxiv2022

Genetic regulation of human aortic smooth muscle cell gene expression and splicing predict causal coronary artery disease genes

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies (GWAS) have identified over 175 loci associated with CAD. The majority of these loci are in non-coding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we hypothesized that a subset of the CAD GWAS risk loci are associated with the regulation of transcription in distinct SMC phenotypes. Here, we measured gene expression in SMCs isolated from the ascending aortas of 151 ethnically diverse heart transplant donors in quiescent or proliferative conditions and calculated the association of their expression and splicing with [~]6.3 million imputed single nucleotide polymorphism (SNP) markers across the genome. We identified 4,910 expression and 4,412 splice quantitative trait loci (sQTL) that represent regions of the genome associated with transcript abundance and splicing. 3,660 of the eQTLs had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 of the eQTLs were SMC- and sex-specific, respectively. To identify the effector transcript(s) regulated by CAD GWAS loci, we used four distinct colocalization approaches and identified 84 eQTL and 164 sQTLs that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. Two CAD loci colocalized with a SMC sex-specific eQTL (AL160313.1 and TERF2IP) and another locus colocalized with SMC-specific eQTL (ALKBH8). Also, 27% and 37% of the sQTLs were unique to quiescent or proliferative SMCs, respectively. The most significantly associated CAD locus, 9p21, was an sQTL for the long non-coding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, these results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

genetics↗

bioRxiv2021

Deficiency of ZC3HC1 increases vascular smooth muscle cell migration, proliferation and neointima formation following injury

RationaleThe ZC3HC1 gene has been linked to various cardiovascular traits. One variant, rs11556924-T, has been found to lower the risk of coronary artery disease (CAD) and blood pressure but increases carotid intima-media thickness (IMT). ObjectiveThis study aimed to determine how ZC3HC1 affects IMT using in vitro and in vivo models. We analyzed the effect of the rs11556924-T allele on ZC3HC1 expression in vascular smooth muscle cells (SMCs) from 151 multi-ethnic heart transplant donors. The results showed that rs11556924-T was associated with lower ZC3HC1 expression and faster SMC migration. ZC3HC1 knockdown (KD) experiments supported these findings, showing increased migration and proliferation. Mechanistically ZC3HC1 KD led to decreased expression of contractile marker genes and the accumulation of cyclin B1, a key cell cycle protein. Pathway analysis of differentially expressed genes between ZC3HC1 KD and controls SMCs showed decreased expression of genes in the cell division and cytoskeleton organization pathways, as well as higher expression of genes involved in extracellular matrix organization and cytokine-mediated signaling. To validate these findings in vivo, we generated and characterized knockout (Zc3hc1-/-) mice. These mice had enhanced neointima formation in response to arterial injury and faster SMCs migration ability. However, complete loss of Zc3hc1 led to a significant reduction in SMC proliferation and lower cyclin B1 protein level. In addition, immunostaining and confocal microscopy demonstrated, for the first time, that ZC3HC1 and Cyclin B1 were located at the cleavage furrow during mitotic progression of SMCs. ConclusionsCollectively, our study suggests that lower ZC3HC1/NIPA level leads to increased SMC migration and neointima formation. Moreover, we proposed a biphasic role of NIPA in proliferation. Lower levels of NIPA promote SMC proliferation, while complete loss of NIPA hampers cell division and abrogates proliferation.

genetics↗

bioRxiv2021

Adipocyte-specific modulation of KLF14 expression in mice leads to sex-dependent impacts in adiposity and lipid metabolism

Genome-wide association studies identified single nucleotide polymorphisms on chromosome 7 upstream of KLF14 to be associated with metabolic syndrome traits and increased risk for Type 2 Diabetes (T2D). The associations were more significant in women than in men. The risk allele carriers expressed lower levels of the transcription factor KLF14 in adipose tissues than non-risk allele carriers. To investigate how adipocyte KLF14 regulates metabolic traits in a sex-dependent manner, we characterized high-fat diet fed male and female mice with adipocyte-specific Klf14 deletion or overexpression. Klf14 deletion resulted in increased fat mass in female mice and decreased fat mass in male mice. Female Klf14-deficient mice had overall smaller adipocytes in subcutaneous fat depots but larger adipocytes in parametrial depots, indicating a shift in lipid storage from subcutaneous to visceral fat depots. They had reduced metabolic rates and increased respiratory exchange ratios consistent with increased utilization of carbohydrates as an energy source. Fasting and isoproterenol-induced adipocyte lipolysis was defective in female Klf14-deficient mice and concomitantly adipocyte triglycerides lipase mRNA levels were downregulated. Female Klf14-deficient mice cleared blood triglyceride and NEFA less efficiently than wild type. Finally, adipocyte-specific overexpression of Klf14 resulted in lower total body fat in female but not male mice. Taken together, consistent with human studies, adipocyte KLF14 deficiency in female but not in male mice causes increased adiposity and redistribution of lipid storage from subcutaneous to visceral adipose tissues. Increasing KLF14 abundance in adipocytes of females with obesity and T2D may provide a novel treatment option to alleviate metabolic abnormalities.

physiology↗

bioRxiv2019

Assessing exposure effects on gene expression

In observational genomics datasets, there is often confounding of the effect of an exposure on gene expression. To adjust for confounding when estimating the exposure effect, a common approach involves including potential confounders as covariates with the exposure in a regression model of gene expression. However, when the exposure and confounders interact to influence gene expression, the fitted regression model does not necessarily estimate the overall effect of the exposure. Using inverse probability weighting (IPW) or the parametric g-formula in these instances is straightforward to apply and yields consistent effect estimates. IPW can readily be integrated into a genomics data analysis pipeline with upstream data processing and normalization, while the g-formula can be implemented by making simple alterations to the regression model. The regression, IPW, and g-formula approaches to exposure effect estimation are compared herein using simulations; advantages and disadvantages of each approach are explored. The methods are applied to a case study estimating the effect of current smoking on gene expression in adipose tissue.

genomics↗

bioRxiv2019

Defining data-driven primary transcript annotations with primaryTranscriptAnnotation in R

Nascent transcript measurements derived from run-on sequencing experiments are critical for the investigation of transcriptional mechanisms and regulatory networks. However, conventional gene annotations specify the boundaries of mRNAs, which significantly differ from the boundaries of primary transcripts. Moreover, transcript isoforms with distinct transcription start and end coordinates can vary between cell types. Therefore, new primary transcript annotations are needed to accurately interpret run-on data. We developed the primaryTranscriptAnnotation R package to infer the transcriptional start and termination sites of annotated genes from genomic run-on data. We then used these inferred co-ordinates to annotate transcriptional units identified de novo. Hence, this package provides the novel utility to integrate data-driven primary transcript annotations with transcriptional unit coordinates identified in an unbiased manner. Our analyses demonstrated that this new methodology increases the sensitivity for detecting differentially expressed transcripts and provides more accurate quantification of RNA polymerase pause indices, consistent with the importance of using accurate primary transcript coordinates for interpreting genomic nascent transcription data.\n\nAvailabilityhttps://github.com/WarrenDavidAnderson/genomicsRpackage/tree/master/primaryTranscriptAnnotation

genomics↗

bioRxiv2018

A genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics

Biomechanical cues dynamically control major cellular processes but whether genetic variants actively participate in mechano-sensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies have identified chromosome 1p32.2 as one of the most strongly associated loci with CAD/IS; however, the causal mechanism related to this locus remains unknown. Employing statistical analyses, ATAC-seq, and H3K27ac/H3K4me2 ChIP-Seq in human aortic endothelium (HAEC), our results demonstrate that rs17114036, a common noncoding polymorphism at the 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR/Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional flow by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays further demonstrate that CAD/IS protective allele at rs17114036 in PLPP3 intron 5 confers an increased endothelial enhancer activity. ChIPPCR and luciferase assays show that CAD/IS protective allele at rs17114036 creates a binding site for transcription factor Kruppel-like factor 2, which increases the enhancer activity under unidirectional flow. These results demonstrate for the first time that a human single-nucleotide polymorphism contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cisregulatory elements provide a previously unappreciated layer of regulatory control in cellular mechano-sensing mechanisms.\n\nSignificance StatementBiomechanical stimuli control major cellular functions and play critical roles in the pathogenesis of diverse human diseases. Although recent studies have implicated genetic variation in regulating key biological processes, whether human genetic variants contribute to the cellular mechano-sensing mechanisms remains unclear. This study provides the first line of evidence supporting an underappreciated role of genetic predisposition in cellular mechanotransduction mechanisms. Employing epigenomic profiling, genome-editing, and latest human genetics approaches, our data demonstrate that rs17114036, a common noncoding polymorphism implicated in coronary artery disease and ischemic stroke by genome-wide association studies, dynamically regulates endothelial responses to blood flow (hemodynamics) related to atherosclerosis via regulation of an intronic enhancer. The results provide new molecular insights linking disease-associated genetic variants to cellular mechanobiology.

genomics↗

bioRxiv2018

BART: a transcription factor prediction tool with query gene sets or epigenomic profiles

SummaryIdentification of functional transcription factors that regulate a given gene set is an important problem in gene regulation studies. Conventional approaches for identifying transcription factors, such as DNA sequence motif analysis, are unable to predict functional binding of specific factors and not sensitive to detect factors binding at distal enhancers. Here we present Binding Analysis for Regulation of Transcription (BART), a novel computational method and software package for predicting functional transcription factors that regulate a query gene set or associate with a query genomic profile, based on more than 6,000 existing ChIP-seq datasets for over 400 factors in human or mouse. This method demonstrates the advantage of utilizing publicly available data for functional genomics research.\n\nAvailabilityBART is implemented in Python and available at http://faculty.virginia.edu/zanglab/bart\n\nContact: zang@virginia.edu

bioinformatics↗