Msx Proteins Transactivate the AlphaB-Crystallin Promoter Through the Heat Shock Element

Abstract

Purpose:: Msx genes encode homeodomain transcriptional factors, are important transcriptional regulators for eye development. Based on previous findings from biochemical experiments, Msx proteins were believed to function mainly as transcriptional suppressors. However, gene expression analyses utilizing either overexpression transgenic animals or loss-of-function Msx mutants have shown that Msx genes can suppress and induce gene expression during development. The strongest supporting evidence of the activator model came from our recent finding that Msx can activate Hsp70 expression mediated by Heat transcription factors through the heat shock elements. Therefore, in order to find additional gene targets that can be activated by the Msx, we identified and tested promoters that share similar promoter structure as the Hsp70 promoter. One of these promoters that we identified is the AlphaB-crystallin (Cryab) gene promoter.

Methods:: To determine if Msx1 or Msx2 function as a transcriptional activator on the Cryab promoter, we performed transfection experiments in the C2C12 cell line by co-transfecting luciferase reporter driven by the Cryab promoter together with Msx1 or Msx2 expression plasmids and a LacZ reporter plasmid as an internal transfection control. Twenty six hours later, cells were harvested and their lysates were assayed for beta-galactosidase and luciferase activities using the Dual-Light reporter assay system.

Results:: Co-transfection with Msx leads to enhancement of Cryab promoter activity. Although the activation of Cryab promoter does not require direct binding of Msx proteins to its DNA, the heat shock elements presented on the Cryab promoter are essential for the Msx-dependent activation. We mapped the Msx activation activity to a conserved domain consists of 30 amino acids in the C-termini of Msx1 and Msx2 proteins.

Conclusions:: Msx protein functions as a potent transcriptional activator on the Cryab promoter. Transcriptional activation regulated by Msx is mediated by co-operative interactions between the C-terminal domain of Msx protein and Heat shock factors.

Keywords: transcription factors • genetics • transcription