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K. Kawai, F. Zhuang, III, Y.–H. Liu; Elucidating the Activation Function of Msx Genes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3123.
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Msx genes encode homeodomain transcriptional factors. We have shown that these genes are important regulators of eye development. The molecular mechanisms of Msx–dependent transcriptional regulation are less characterized and much remains to be explored. Several studies have shown that Msx proteins are potent transcriptional suppressors and their suppression activity is mediated by interaction with other protein independent of DNA binding. However, our current study revealed a new function for Msx genes; Msx genes can also function as transcriptional activators.
To determine if Msx1 or Msx2 function as a transcriptional activator of a the Hsp70.1 promoter, we performed transfection experiments by co–transfecting C2C12 myoblast cell line with various Hsp70.1/lacZ reporter constructs together with Msx1 or Msx2 expression plasmids and a luciferase reporter plasmid as an internal transfection control. Thirtysix hours later, cells were harvested and their lysates were assayed for beta–galactosidase and luciferase activities using the Dual–Light reporter assay system.
HSP70.1 promoter was widely used to as the basal promoter for studying enhancer activity in transgenic mice. Recent studies have shown that HSP70.1 is one of the target genes of Cyclin D1. Msx1 was shown to activate cyclinD1 expression as a strategy to maintain cells in undifferentiated state by preventing cells from exiting cell cycle. In order to understand the underlying molecular mechanism of Msx–dependent gene activation, we examined the transcriptional response of a 650bp DNA fragment of the Hsp70.1 promoter to Msx1 or Msx2. The 650bp Hsp70.1 promoter responded to Msx transactivation in a gene dosage dependent manner. Further deletions of the 650bp Hsp70.1 promoter allowed us to determine that The Heat Shock Element (HSE) as well as a NF–kappa B binding site within 210 bases 5’ of the transcriptional start site on the Hsp70 promoter is required for transcriptional activation by the Msx. However, DNA binding by Msx is not required for this activation. We mapped the Msx activation activity to a conserved domain consisted of 30 amino acids in the C–termini of Msx1 and Msx2 proteins.
Msx can activate Hsp70 transcription without its DNA binding activity, and the activation activity is dependent on a conserved domain consisted of 30 amino acids in its C–terminus. Our working model is that Msx may modify other transcriptional factor(s) to enhance the expression of Hsp70.1 or other downstream targets.
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