Search

Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Myocardin (MYOCD) is an essential component of a molecular switch for the expression of contractile genes in smooth muscle and cardiac muscle cells. The Myocd gene comprises at least fifteen exons, including two alternately spliced exons designated 2a and 10a. We investigated tissue-specific Myocd expression in mouse, rat and human tissues to determine the conservation in expression of each Myocd splice variant and to ascertain whether any functional differences exist among MYOCD isoforms. Conventional and quantitative RT-PCR revealed the dominant expression of Myocd exon 2a (Myocd_v3) in smooth muscle cell (SMC)-rich tissues (aorta and bladder) with little expression in heart across all species studied. Each species of heart showed primarily a longer version of Myocd (Myocd_v1) without exon 2a. While exclusion of exon 2a was common in all cardiac muscle samples, exon skipping of Myocd exon 10a was a rare event in both cardiac muscle and SMC tissues. In general, all four MYOCD isoforms showed comparable stimulation of SMC promoters. On the other hand, Myocd_v1 and Myocd_v2 were more active than Myocd_v3 and Myocd_v4 in stimulating cardiac muscle promoters and Myocd_v1's activity was augmented in the presence of the cardiac transcription factor, MEF2C. Importantly, whereas all four MYOCD isoforms similarly induced expression of endogenous SMC genes in a prostate tumor cell line (LNCaP), none could induce the endogenous expression of specific cardiac markers. These results are the first to show relative expression and activities of the major myocardin isoforms across disparate species. We propose a new myocardin nomenclature reflecting the dominant splice variants expressed in cardiac muscle (Myocd_v1 and v2) versus SMC-rich tissues (Myocd_v3 and v4)., (Copyright 2010 Elsevier B.V. All rights reserved.)

Smooth muscle cells (SMC) express a battery of cell-restricted differentiation genes, many of which are down-regulated during the course of vascular disease. Here, we present the mRNA expression, genomic structure and chromosomal mapping of the gene encoding human smooth muscle cell calponin (SMCC). Human SMCC transcripts are restricted to tissues and cells of SMC origin and, in the latter case, appear to be uniquely controlled in two distinct human SMC lines of uterine and aortic origin. Restriction mapping. Southern blot and PCR analysis of a 70-kb human bacterial artificial chromosome (BAC) revealed a genomic structure (seven exons spanning > 11 kb) very similar to that reported for the mouse SMCC gene. Using a variety of human-rodent somatic cell hybrid and radiation hybrid mapping panels, the human SMCC gene was mapped to a genomic interval of less than 1.32 Mb in 19p13.2. These results provide new information concerning the regulation of SMCC gene expression and demonstrate the utility of two human SMC lines for the further characterization of this gene's expression control. The identification of a BAC harboring the entire human SMCC locus represents an important reagent for future analysis of SMCC regulatory sequences. Finally, the localization of SMCC to a defined genomic interval will facilitate an analysis of its potential as a candidate gene for disease phenotypes mapping to 19p13.2.