Abstract
Previous studies in our laboratory demonstrated that the transgene consisting of the -4.2 to +11.6 kilobase (kb) region of the smooth muscle (SM) myosin heavy chain (MHC) gene was expressed in virtually all SM tissue types in vivo in transgenic mice and that the multiple CArG elements within this region were differentially required in SMC subtypes, implying that the SM-MHC gene was controlled by multiple transcriptional regulatory modules. To investigate this hypothesis, we analyzed specific regulatory regions within the SM-MHC -4.2 to +11.6 kb region by a combination of deletion analyses of various SM-MHC transgenes as well as by DNaseI hypersensitivity assays and in vivo footprinting in intact SMC tissues. The results showed that SM-MHC transgene expression depended on a large number of required regulatory modules that were widely spread over the -4.2 to +11.6 region. Moreover, the results revealed several unexpected novel features of regulation of the SM-MHC gene including: 1) unique combinations of regulatory modules were required for SM-MHC expression in different SMC-subtypes; 2) repressor modules as well as activator modules were both critical for SMC specificity of the gene; 3) certain modules were required in certain contexts but were dispensable in others within a given SMC-subtype (i.e. the net activity of the module was determined by interaction between modules not simply by the sum of module activities); and 4) we identified a highly conserved 200-base pair transcriptional regulatory module at +8 kb that was required in the large arteries but dispensable in the coronary arteries and airways in transgenic mice and contained multiple potential cis-elements that were occupied by nuclear proteins in the intact aorta based on in vivo footprinting. Taken together, the results suggest a model of complex modular control of expression of the SM-MHC gene that varies between SMC subtypes. Moreover, the studies establish the possibility of designing derivatives of the SM-MHC promoter that might be used for targeting gene expression to specific SMC subtypes in vivo.
Highlights
Smooth muscle (SM)1 myosin heavy chain (MHC) is one of the best markers for smooth muscle cell (SMC) lineages [1]
Previous studies in our laboratory demonstrated that the transgene consisting of the ؊4.2 to ؉11.6 kilobase region of the smooth muscle (SM) myosin heavy chain (MHC) gene was expressed in virtually all SM tissue types in vivo in transgenic mice and that the multiple CArG elements within this region were differentially required in SMC subtypes, implying that the SMMHC gene was controlled by multiple transcriptional regulatory modules
Given the very large size of the SM-MHC regulatory region (ϳ16 kb), we employed multiple experimental methods and strategies to characterize regulatory regions including 1) DNaseI hypersensitive assays and in vivo footprinting to identify regions that may contain transcriptionally active elements based on evidence of binding of nuclear proteins, 2) identification of regions that are conserved across species, 3) testing various SM-MHC promoter constructs based on transient transfection studies in cultured SMCs, and 4) testing selected promoter constructs in transgenic mice
Summary
Smooth muscle (SM) myosin heavy chain (MHC) is one of the best markers for smooth muscle cell (SMC) lineages [1]. As a first step to elucidate the transcriptional regulatory mechanisms of the SM-MHC gene, we previously analyzed the function of three conserved CArG elements within the 5Ј-flanking and first intronic sequences in transgenic mice [6] Results of these studies demonstrated that the three CArG elements were differentially required in SMC subtypes in vivo. These results implied that expression of the SM-MHC gene was regulated by multiple transcriptional regulatory regions including the 5Ј-flanking and intronic regions containing the CArG elements These studies examined the function of individual cis-elements and did not define the precise regulatory regions within the Ϫ4.2 to ϩ11.6 SM-MHC region required for SMC-specific gene expression in vivo. These results imply that the SM-MHC gene regulatory system consists of multiple modular regulatory domains that confer the capability for SMCs to respond to vastly divergent environmental cues in developmental space and time and under pathophysiological conditions
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