| Abstract
| - Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions duringcraniofacial skeletal and neural development. To identify coregulatory molecules that participate intranscriptional repression by Msx2, we applied a Farwestern expression cloning strategy to identifytranscripts encoding proteins that bind Msx2. A λgt11 expression library from mouse brain was screenedwith radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNAwas isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein bindingactivity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment,and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On thebasis of biochemical analyses, this novel protein was named MINT, an acronym for Msx2-interactingnuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testisand at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis librarywere sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met in goodKozak context, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open readingframe with the Msx2 interaction domain residues 2070−2394. Protein sequence analysis reveals thatMINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Westernblot analysis of fractionated cell extracts reveals that mature ∼110 kDa (N-terminal) and ∼250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregatingwith Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- andG-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal ratosteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINTand OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts.Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1−812) suppresses the FGF/forskolin-activated OC promoter, does not significantly regulate CMVpromoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. Intoto, these data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulatesOC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specificregulatory region of the OC proximal promoter, nucleotides −141 to −111. The N-terminal MINT RRMdomain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein.Acting as a scaffold protein, MINT potentially exerts both positive and negative regulatory actions byorganizing transcriptional complexes in the nuclear matrix.
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