biostudies-arrayexpressDanielle Charissa DyHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15722TWIST1, a bHLH transcription factor, regulates mesenchymal specification, differentiation, proliferation and migration during development and in diseases such as cancer. More recently, genome-wide association studies have identified TWIST1 as a causal gene that increases risk for multiple vascular diseases, including atherosclerosis and hypertension. However, its molecular role in the vascular wall remains unclear. In this study, we interrogated how TWIST1 dimer composition and discrete TWIST1 domains affect SMC phenotype by expressing forced TWIST1 dimers or TWIST1 variants lacking specific domains, followed by bulk RNA sequencing and proliferation and migration assays in human coronary artery SMCs (HCASMCs). We found that TWIST1 homodimers had only modest transcriptomic effects but strongly promoted migration and proliferation - effects abolished by deletion of the TWIST1 N-terminus. Heterodimerization of TWIST1 with TCF3-endoded E proteins resulted in larger transcriptomic effects, promoting Rho/ROCK signaling and extracellular matrix production/organization, but had only modest effects on proliferation and no effect on migration. Deletion of the TWIST1 C-terminus resulted in a very large transcriptomic shift with predicted downregulation of angiotensin and Rho/ROCK signaling as well as ECM production/organization pathways, in a manner suggesting a dominant-negative effect on TWIST1-E12 function. Comparison with single-cell RNA-seq data from human endarterectomy samples placed the function of these TWIST1 variants in a disease context and showed that deletion of the C-terminal domain prevented a modulated SMC phenotype. These studies demonstrate that TWIST1 influences different aspects of SMC phenotype independently via discrete domains and dimer composition, and link TWIST1 to key signaling pathways that influence SMC phenotype during disease.biostudies-arrayexpressSample Collection - Human coronary artery smooth muscle cells (HCAMSCs) were ordered from Lonza. HCAMSCs were transduced with lentivirus and prepared for RNA isolation.Library Construction - Following quality control, the NEBNext Ultra II Directional Kit was used for library preparation.Growth Protocol - HCAMSCs were cultured in Lonza's SmBM medium with SmGM-2 Smooth Muscle Cell Growth Medium-2 Bullet Kit, excluding gentamicin sulfate. HCAMSCs were used between passage 5-6 for all experiments.Sample Treatment - Once HCAMSCs reached 70% confluence in 6-well plates, serum-free media was applied for 24 hours prior to lentiviral transduction with polybrene. After overnight incubation, the medium was replaced with fresh full-serum SmGM medium. HCASMCs were harvested for RNA isolaion 48 hours later.Sequencing - Libraries were sequenced using the NovaSeq X Plus platform.Nucleic Acid Extraction - RNA extraction was performed using the Zymo Quick-RNA Microprep Kit.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - For Principal Component Analysis of samples, the Transcripts Per Million (TPM) values were normalized using a log2 transformation. Normalized TPM values were also used to perform and visualize Pearson’s Correlation and hierarchical clustering analyses. Differential gene expression analysis was performed using the expected counts of each sample.Sequence Alignment - The reference genome GRCh38.primary_assembly reference 204 genome was used. Reads were trimmed using fastp (v0.23.4) and alignment was 205 performed using STAR (v2.7.10a) and the count matrix data were generated using 206 RSEM (v1.3.1) with a minimum mapped reads value of 5.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq X<h4>Introduction</h4>TWIST1, a bHLH transcription factor, regulates mesenchymal specification, differentiation, proliferation and migration during development and in diseases such as cancer. More recently, genome-wide association studies have identified TWIST1 as a causal gene that increases risk for multiple vascular diseases, including atherosclerosis and hypertension. However, its molecular role in the vascular wall remains unclear.<h4>Methods</h4>In this study, we interrogated how TWIST1 dimer composition and discrete TWIST1 domains affect SMC phenotype by expressing forced TWIST1 dimers or TWIST1 variants lacking specific domains, followed by bulk RNA sequencing and proliferation and migration assays in human coronary artery SMCs (HCASMCs).<h4>Results</h4>We found that TWIST1 homodimers had only modest transcriptomic effects but strongly promoted migration and proliferation-effects abolished by deletion of the TWIST1 N-terminus. Heterodimerization of TWIST1 with TCF3-encoded E proteins resulted in larger transcriptomic effects, promoting Rho/ROCK signaling and extracellular matrix production/organization, but had only modest effects on proliferation and no effect on migration. Deletion of the TWIST1 C-terminus resulted in a very large transcriptomic shift with predicted downregulation of angiotensin and Rho/ROCK signaling as well as ECM production/organization pathways, in a manner suggesting a dominant negative effect on TWIST1-E12 function. Comparison with single-cell RNA-seq data from human endarterectomy samples placed the function of these TWIST1 variants in a disease context and showed that deletion of the C-terminal domain prevented a modulated SMC phenotype.<h4>Discussion</h4>These studies demonstrate that TWIST1 influences different aspects of SMC phenotype independently via discrete domains and dimer composition, and link TWIST1 to key signaling pathways that influence SMC phenotype during disease.RNA-seq of coding RNAHomo sapiensFunctional analysis of TWIST1 domains regulating smooth muscle cell phenotypeDanielle Charissa DyRobert WirkaDanielle Charissa Maramara Dy, Thiel Lehman, Adrian Othon, Mitesh Rathod, William J Polacheck, Robert WirkafalseFunctional analysis of TWIST1 domains regulating smooth muscle cell phenotypeTWIST1, a bHLH transcription factor, regulates mesenchymal specification, differentiation, proliferation and migration during development and in diseases such as cancer. More recently, genome-wide association studies have identified TWIST1 as a causal gene that increases risk for multiple vascular diseases, including atherosclerosis and hypertension. However, its molecular role in the vascular wall remains unclear. In this study, we interrogated how TWIST1 dimer composition and discrete TWIST1 domains affect SMC phenotype by expressing forced TWIST1 dimers or TWIST1 variants lacking specific domains, followed by bulk RNA sequencing and proliferation and migration assays in human coronary artery SMCs (HCASMCs). We found that TWIST1 homodimers had only modest transcriptomic effects but strongly promoted migration and proliferation - effects abolished by deletion of the TWIST1 N-terminus. Heterodimerization of TWIST1 with TCF3-endoded E proteins resulted in larger transcriptomic effects, promoting Rho/ROCK signaling and extracellular matrix production/organization, but had only modest effects on proliferation and no effect on migration. Deletion of the TWIST1 C-terminus resulted in a very large transcriptomic shift with predicted downregulation of angiotensin and Rho/ROCK signaling as well as ECM production/organization pathways, in a manner suggesting a dominant-negative effect on TWIST1-E12 function. Comparison with single-cell RNA-seq data from human endarterectomy samples placed the function of these TWIST1 variants in a disease context and showed that deletion of the C-terminal domain prevented a modulated SMC phenotype. These studies demonstrate that TWIST1 influences different aspects of SMC phenotype independently via discrete domains and dimer composition, and link TWIST1 to key signaling pathways that influence SMC phenotype during disease.2025-10-14T00:00:00Z2026-05-27T14:28:27.316Z2025-10-14T22:49:09.851ZE-MTAB-15722ERP182173EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.3389/fcvm.2025.1659847