There is now incontrovertible evidence that vascular smooth muscle cells (VSMCs) contribute substantively to vascular diseases. The function or dysfunction of VSMCs is driven, in part, by the activity of key transcription factors (TF). Serum response factor (SRF), an abundantly expressed TF in VSMCs that binds a large cadre of CArG boxes colloquially known as the CArGome, orchestrates a number of disparate gene programs. Surprisingly, almost nothing is known about the in vivo regulation of Srf transcription and, while the function of SRF in vascular development is well understood, there is no information about its direct role in vascular diseases; and gender-based studies are not possible given the limitation of the most popular SMC Cre driver (Myh11). Moreover, the full complement of SRF target genes (notably long noncoding RNAs, lncRNAs) is not known. We have been a leading lab in SRF research and now offer fresh insights into these major scientific gaps that, collectively, form the basis of this application. First, we provide CRISPR-Cas9 genome editing results, bioinformatic predictions, ChIP-seq, and luciferase data supporting functional transcription factor bind- ing sites (TFBS) controlling Srf transcription in vivo. Second, gene expression and Srf loss-of-function (LOF) studies support SRF as an early mediator of VSMC growth, inflammation, and neointimal formation following acute vascular insult. Importantly, existing Cre driver mice limit analysis of Srf LOF to a narrow time window of only 10-14 days post-tamoxifen due to a competing, lethal phenotype of the gastrointestinal (GI) tract. How- ever, we have recently generated and validated a new Cre driver mouse with restricted Cre-mediated excision to VSMCs; little activity is observed in visceral SMCs of the GI tract, and a cross with floxed Srf mice shows extended survival providing the first ever opportunity to interrogate the function of SRF in both acute and chronic models of vascular disease without confounding phenotypes. Finally, genomic studies implicate a new SRF-dependent VSMC inflammatory gene program and CRISPR studies show a specific base substitution within the CArG box nullifies SRF-dependent gene expression in vivo. Three integrated aims will rigorously test the hypothesis that multiple TFBS control Srf expression to direct CArG-dependent homeostatic or pathogenic gene programs in the vessel wall. Aim 1 will evaluate the function of new TFBS governing Srf transcription using CRISPR editing in the mouse. Aim 2 will elucidate phenotypes associated with Srf LOF and gain-of-function in acute and chronic models of vascular disease using a novel Cre driver mouse for unparalleled VSMC specificity. Aim 3 will further utilize mice in Aim 2 for integrative VSMC ChIP-seq and RNA- seq studies to elucidate novel SRF target genes, particularly the class of lncRNAs, in the control of VSMC phenotypes; CRISPR editing of key CArG boxes are planned as a new paradigm to study gene function. These studies will vertically advance our knowledge of SRF regulation and function, paving the way towards new therapeutic approaches to combat vascular diseases while advancing new directives for further research.
- National Institutes of Health: $533,901.00