Function and Transcriptional Regulation of Leiomodin1

PROJECT SUMMARY Recent lineage tracing and single cell omics studies provide irrefutable evidence for vascular smooth muscle cell (VSMC) state and fate changes that contribute to vascular disease. Leiomodin1 (LMOD1), a VSMC marker and known GWAS risk allele for human coronary artery disease (CAD), is a cytoskeletal gene whose in vivo function and regulation are completely unknown. Previous work from our lab showed Lmod1 to be a direct target of the SRF/Myocardin transcriptional switch. We also discovered that genetic loss of Lmod1 results in a lethal neonatal visceral myopathy in both humans and mice, precluding any assessment of postnatal VSMCs. New floxed Lmod1 mice rapidly succumb to a similar visceral myopathy with the Myh11-CreERT2 mouse; however, conditional loss of Lmod1 with a new VSMC-restricted Itga8-CreERT2 mouse is compatible with life. Significantly, mutant mice show a CAD phenotype and death following a PCSK9-high fat diet. Preliminary studies with spatial transcriptomics and immunofluorescence/electron microscopy support the dedifferentiation of Lmod1 null coronary VSMCs. Further studies demonstrate nuclear localization of LMOD1 and its interaction with MYBBP1A, a transcription factor involved in ribosomal RNA biogenesis. Loss- and gain-of-function studies support a homeostatic function of LMOD1 in maintaining normal MYBBP1A-dependent ribosomal biogenesis in VSMCs. Finally, CRISPR-mediated deletion of an intronic enhancer, encompassing an SRF-binding CArG box and closely juxtaposed FOXO3 element (site of CAD-associated single nucleotide variant [SNV]), reduces Lmod1 expression in vivo. These preliminary findings support the hypothesis that SRF/FOXO3-dependent Lmod1 transcription safeguards coronary VSMCs by maintaining normal MYBBP1A activity. Aim 1 will elucidate the nature of the CAD phenotype using mouse genetics, spatial profiling, and immuno-electron microscopy. Aim 2 will elucidate nuclear LMOD1-MYBBP1A biology using structure-function studies, click chemistry for RNA biogenesis, and a new floxed Mybbp1a mouse that will directly test its role in VSMC RNA biogenesis and CAD. Aim 3 will elucidate the transcriptional control of Lmod1 in vivo through chromosome conformation capture and Prime editing of CArG and FOXO3 sites to assess individual and combined effects on CAD. These studies, which constitute the first in vivo interrogation of LMOD1 function and gene regulation, will uncover a novel nuclear function of a VSMC-restricted CAD risk allele, affording insight into its role in maintaining normal coronary artery homeostasis. Such information will provide a foundation for developing potential clinical interventions that preserve LMOD1 levels and/or the LMOD1-MYBBP1A association as a means of mitigating CAD risk in humans.