Abstract 463: The Key Regulatory Elements for SM22 Transcription

Abstract

Gene transcription is controlled by an array of transcriptional regulatory elements. SM22 gene regulation has been widely used to characterize the molecular mechanisms of smooth muscle cell (SMC) phenotypic modulation during cardiovascular development and in vascular diseases. Our previous studies show that the proximal CArG box (CArGnear) in the SM22 promoter is required for its transcription in arterial smooth muscle cells (SMC). However, the role of the CArG box in visceral SMCs has not yet been explored. Here we aim to determine the role of CArG boxes in regulating S M22 transcription in both vascular and visceral SMCs. Using bacterial chromosome (BAC) recombineering, we knock-in a lacZ reporter into a SM22 BAC to trace SM22 transcriptional activities in transgenic mice. Anatomic/histology analyses show that the lacZ expression patterns in the BAC transgenic mice recapitulate that of the endogenous SM22 transcription during embryogenesis and in adult . Similar to the endogenous SM22 regulation, the expression of lacZ is highly sensitive to vascular remodeling: carotid injury abolishes lacZ expression in the arterial wall. Using seamless BAC recombineering mutagenesis, we generate mutations in the proximal and/or distal CArG box in the SM22-lacZ-BAC. Consistent with our previous results, we find that mutating the CArGnear box disrupts the lacZ expression in the aorta; this mutation also drastically reduces its expression in visceral SMCs including stomach, uterus and bladder. Interestingly, mutating the distal CArG (CArGfar) box does not affect the lacZ expression in arterial, venous and visceral SMCs. Mutating both CArG boxes nearly abolishes lacZ expression in all SMCs. This study provides evidence supporting the generation of SM22 knockout mice by mutating the CArG boxes in the SM22 promoter using the CRISPR technology.