Cryptic MCAT Enhancer Regulation in Fibroblasts and Smooth Muscle Cells

Abstract

An asymmetric polypurine-polypyrimidine cis-element located in the 5 region of the mouse vascular smooth muscle ␣-actin gene serves as a binding site for multiple proteins with specific affinity for either singleor double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to transactivate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur␣ and Pur␤ with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur␣, Pur␤, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur␣, Pur␤, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur␣, Pur␤, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.Current models of transcriptional repression take into account the ability of negative-acting factors to function in multiple capacities with or without DNA binding specificity (1, 2). For example, evidence exists that certain activator proteins can simply be masked or sequestered by protein binding partners that do not interact with DNA directly, as in the case of retinoblastoma tumor suppressor protein-mediated repression of the E2F family of trans-activators (3). Alternatively, a repressor protein may bind and displace components of the basal transcription machinery as in the case of Drosophila evenskipped-mediated repression of the Adh proximal promoter (4). Recent work suggests that some repressors can participate, indirectly, in modifying chromatin structure by binding activator sites and recruiting specific histone deacetylases to silence genes through histone modification. This scenario is implicated in repression of E-box motifs through Mad/Max-mediated recruitment of histone deacetylases 1 and 2 (2). Still other models of repression propose that activator protein access to a DNA target sequence can be blocked by repressor protein binding to either the same site or an overlapping site. Such a mechanism appears to be operative in the competitive DNA binding of YY1 and serum response factor (SRF) 1 to serum response elements (SREs) of the c-fos promoter (5).An interesting variation on the theme of multiple proteins competing for overlapping binding sites arises from our attempts to elucidate the mechanism of vascular smooth muscle (VSM) ␣-actin pr...

Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Purβ

“…Although the molecular weight of N-HisPur␤ calculated on the basis of its amino acid sequence is 35,168.6, it appears to migrate by SDS-PAGE as an ϳ43-kDa peptide under reducing conditions. The unusual electrophoretic mobility of the recombinant protein is consistent with the reported mobility of native Pur␤ expressed in fibroblasts and vascular smooth muscle cells (13,23). This suggests that the His tag is not the major contributing factor to the non-ideal electrophoretic behavior of N-HisPur␤.…”

“…The most significant sequence differences between Pur␣ and Pur␤ exist near the N and C termini, suggesting that each protein may have evolved to perform distinct functions (13). Pur␣ and Pur␤ have been reported to bind to a PUR element in a highly asymmetric polypurine/polypyrimidine tract located in the 5Ј-flanking region of the mouse SM␣A gene (22,23). It has been hypothesized that strand-specific binding by Pur␣/Pur␤ to this element disrupts a core MCAT enhancer motif, thereby repressing SM␣A promoter activity in cultured fibroblasts and vascular smooth muscle cells (23).…”

“…Pur␣ and Pur␤ have been reported to bind to a PUR element in a highly asymmetric polypurine/polypyrimidine tract located in the 5Ј-flanking region of the mouse SM␣A gene (22,23). It has been hypothesized that strand-specific binding by Pur␣/Pur␤ to this element disrupts a core MCAT enhancer motif, thereby repressing SM␣A promoter activity in cultured fibroblasts and vascular smooth muscle cells (23). That Pur␣ and Pur␤ function as inhibitors of SM␣A expression is of particular interest due to the essential role played by SM␣A in vascular contraction (24), cell motility (25), wound repair (26), and arterial remodeling (27,28).…”

Single-stranded DNA-binding Proteins PURα and PURβ Bind to a Purine-rich Negative Regulatory Element of the α-Myosin Heavy Chain Gene and Control Transcriptional and Translational Regulation of the Gene Expression

“…It is of interest to note that PUR proteins have been shown to bind to other cardiac myogenic factors, e.g. TEF-1 and SRF, which also play prominent roles in ␣-MHC gene expression (14,32). Recently, YY1 has been shown to bind to an upstream element in the human ␣-MHC gene promoter where it negatively regulates the gene expression (51).…”

“…Earlier reports have shown a role of PUR␣/␤ proteins in smooth muscle-specific expression of the vascular ␣-actin gene. Kelm and co-workers (32) show that PUR proteins bind to the plus strand of a cryptic M-CAT element of the vascular ␣-actin gene and cooperate with TEF-1 and another single strand DNA-binding protein, MSY1, which binds to the FIG. 6.…”