1: Mol Cell Biochem. 2004 Dec;267(1-2):1-11. The mechanism of biogenesis and potential function of the two alternatively spliced mRNAs encoded by the murine Msx3 gene. Matsui H, Takahashi T, Raghow R. Departments of Pharmacology, Pediatrics and Biomedical Engineering, University of Tennessee Health Science Center, Memphis, TN, USA. The homeodomain-containing Msx3 gene, the newest member of the Msx family, encodes two mRNAs, with an unknown relationship to each other. To elucidate how Msx3 gene generates the two transcripts, we cloned their corresponding cDNAs from an E10.5 mouse embryo cDNA library. The alignment of sequences of the two Msx3-specific cDNAs with the corresponding regions of the genomic DNA revealed that read-through of the sequences preceding a cryptic splice donor site in the first intron of Max3 gene generated the longer transcript. The longer Msx3 mRNA (Msx3-l) contains 66 nucleotides spliced in frame that would encode a protein with 22 additional amino acids. These extra 22 amino acids are inserted between the residues 72 and 73, exactly 14 amino acids upstream of the homeodomain of the smaller Msx3 protein. In situ hybridization and competitive RT-PCR experiments revealed that both Msx3-s and Msx3-l mRNAs elicited similar spatio-temporal patterns of expression in the developing embryo, with maximal expression of both mRNAs occurring in the embryos between 8.5 and 12.5 days post-coitus. We found that while Msx3-s down-regulated Msx1 promoter in transfected C2C12 cells, co-expression of Msx3-l alone did not affect the activity of the Msx1 promoter. In contrast, a concomitant expression of Msx3-s and Msx3-l in the transfected C2C12 cells neutralized the repressive effect of Msx3-s on the Msx1 promoter. In transient expression assays, the repressive action of Msx3-s on Msx1 promoter could also be reversed by co-expression of exogenous Sp1. Our data indicate that the potential interactions among the protein products encoded by the alternately spliced Msx3 mRNAs and the putative constituents of transcriptional co-activators and co-repressors may have functional consequences in vivo. PMID: 15663180 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Biochem J. 1999 May 1;339 ( Pt 3):751-8. Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300). Shetty S, Takahashi T, Matsui H, Ayengar R, Raghow R. Department of Biology, The University of Memphis, Memphis, TN 38152, USA. The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We demonstrate that Msx1-mediated autorepression remained unaffected, regardless of the presence or absence of the Msx1 recognition motifs on the promoter. Furthermore, graded exogenous expression of TATA-binding protein (TBP), Sp1 or cAMP-response-element-binding protein-binding protein (CBP/p300) could counteract the autoinhibitory activity of Msx1. Finally, we demonstrate that Msx1 protein can be immunoprecipitated in a multiprotein complex containing TBP, Sp1 and CBP/p300. We hypothesize that the interaction of Msx1 protein with one or more ubiquitous or tissue-restricted transcription factors mediates transcriptional autorepression of the Msx1 gene. PMID: 10215616 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Biol Chem. 1997 Sep 5;272(36):22667-78. A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice. Takahashi T, Guron C, Shetty S, Matsui H, Raghow R. Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163, USA. 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 the Msx-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 minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-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 the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell 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 beta-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-1 promoter 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. PMID: 9278425 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------