Abstract 985: BAC Transgenic Analysis of the Smooth Muscle Calponin Gene
Regulatory elements controlling gene expression are often found in separate, sometimes remote, regions around gene loci. Artificial chromosomes offer a means to capture all regulatory elements for study of gene regulation in a near-correct genomic context. The smooth muscle calponin gene (CNN1) encodes for a multifunctional protein involved in signaling, contractile force generation, and growth regulation. While CNN1’s physiology has been studied extensively, its transcriptional regulation has proven to be intractable to conventional in vivo assays. Four evolutionarily-conserved serum response factor (SRF) binding sites (called CArG boxes) are present in the first intron of CNN1 and appear to be required for full transcriptional competence of CNN1 in cultured smooth muscle cells (SMC). To assess the functionality of CNN1 CArG elements in vivo, we exploited a 103-kb bacterial artificial chromosome (BAC) containing the human CNN1 locus shown previously to completely recapitulate endogenous mouse CNN1 expression in vivo. We now show similar SMC-restricted expression of human CNN1 from only 38-kb of BAC sequence. BAC recombineering studies in which the first intron (containing four CArG elements) of CNN1 is replaced with a bacterial selection gene (galK) reveal complete loss of human CNN1 expression in vascular SMC, with residual expression in some visceral SMC-containing tissues such as bladder. Transgenic mice carrying the 38-kb BAC with point mutations in all four intronic CArG elements show similar findings suggesting that SRF directly mediates in vivo expression of CNN1 primarily through intronic CArG elements. Bioinformatic, luciferase, and ChIP assays reveal the presence of distal, conserved CArG elements, including a previously unrecognized one located more than 3-kb upstream of the CNN1 transcription start site. The latter CArG-like element is responsive to the SRF coactivator myocardin and is essential for full CNN1 promoter activity in vitro. Moreover, this distal CArG-like element is embedded in a cis regulatory module with conserved binding sites to other distinct transcription factors. We propose that full in vivo CNN1 gene expression requires multiple elements, including five CArG boxes, spread over 14-kb of genomic sequence.