A Minimal Murine Msx-1 Gene Promoter

Abstract

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from −1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C₂C₁₂ cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5′ deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of theMsx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C₂C₁₂ cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimalMsx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with thecis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5′-flanking regions from −161 to −154 and from −26 to −13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of theMsx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophilacell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing −165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed β-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.