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Published on: February 15, 2022
Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum
Manabu Nagao1, Qing Lyu2, Quanyi Zhao1
1From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.N., Q.Z., R.C.W., J.B., T.N., P.C., J.B.K., M.P., T.Q.).
Rationale:
The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known.
Objective:
To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway.
Methods And Results:
Modulation of TCF21 expression in human coronary artery SMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation-sequencing to map SRF-binding sites in human coronary artery SMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at colocalized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein coimmunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction.
Conclusions:
These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this coronary artery disease-associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in coronary artery disease risk.
Insights
Transcription factor 21 (TCF21) antagonizes the myocardin-serum response factor (MYOCD-SRF) pathway, revealing its role in smooth muscle cell phenotype and coronary artery disease risk.
Area of Science:
- Cardiovascular Biology
- Molecular Genetics
- Vascular Cell Biology
Background:
- Genome-wide association studies link TCF21 to coronary artery disease (CAD) risk.
- TCF21 influences smooth muscle cell (SMC) phenotype in murine atherosclerosis models.
- Human TCF21 expression is associated with reduced CAD risk, but its mechanism is unknown.
Purpose of the Study:
- Investigate the molecular mechanisms by which TCF21 regulates SMC phenotype.
- Determine if TCF21 affects the MYOCD-SRF pathway, crucial for SMC lineage.
Main Methods:
- Assessed TCF21 effects on SMC markers and MYOCD/SRF in human coronary artery SMC.
- Performed ChIP-sequencing to map SRF-binding sites and TCF21 colocalization.
- Utilized in vitro genome editing and reporter assays to study regulatory elements.
- Conducted co-immunoprecipitation to examine protein interactions.
Main Results:
- TCF21 suppresses SMC markers and MYOCD/SRF transcription factors at RNA and protein levels.
- TCF21 colocalizes with SRF-binding sites, including a novel SRF enhancer and the MYOCD promoter.
- Genome editing confirmed SRF enhancer activity and its regulation by TCF21.
- TCF21 directly interacts with MYOCD, blocking MYOCD-SRF complex formation.
Conclusions:
- TCF21 antagonizes the MYOCD-SRF pathway via multiple mechanisms.
- Establishes TCF21's role in SMC processes and phenotypic modulation.
- Highlights the significance of SMC response to vascular stress in CAD risk.
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