CRISPR Editing of the SRF-Myocardin Transcriptional Switch

Vascular smooth muscle cell (VSMC) differentiation is defined by the expression of cyto-contractile genes that confer quiescence and contractile activity. The major effector of VSMC differentiation is the serum response factor/myocardin (SRF/MYOCD) transcrip-tional switch, which binds CArG boxes and induces expression of many VSMC-restricted genes. We know essentially nothing about the non-coding sequences controlling this switch in vivo. Preliminary studies in mice have begun to elucidate the expression of MYOCD protein and the importance of key non-coding sequences upstream and downstream of the SRF/MYOCD switch. Using CRISPR-Cas9 genome editing and state-of-the-art tools in genomics, we propose three aims to test the hypothesis that transcription of MYOCD, and downstream target genes, proceed in an SRF-dependent and SRF-independent manner. Aim 1 will utilize a new Myocd-FLAG mouse model to track, for the very first time, the endogenous expression of MYOCD protein during embryonic and post-natal development. Aim 2 will make further use of the Myocd-FLAG mouse to perform a series of CRISPR-Cas9 genome edits of known and newly defined enhancers and regulatory elements derived from ChIP-seq, computational prediction, and circular chromosome conformation capture experiments. Aim 3 will then turn to the definition of the MYOCARDome in VSMC using an integrative genomics approach. Existing and planned RNA-seq/ChIP-seq studies will be intersected to define both SRF-dependent and SRF-independent target genes (coding and noncoding) of MYOCD in an in vivo setting using the Myocd-FLAG mouse and a new inducible, HA-tagged Myocd mouse line we have generated and validated. Completion of the aims will vertically advance our understanding of VSMC differentiation during embryonic and postnatal development. Further, fresh insight will be gained into the regulatory processes undergirding dynamic Myocd expression under normal and stress-induced states where the program of VSMC differentiation is often compromised leading to VSMC diseases. (AHA Program: Grant-in-Aid)