Cell-specific activation of the atrial natriuretic factor promoter by PITX2 and MEF2A.
The PITX2 homeodomain protein is mutated in patients with Axenfeld-Rieger syndrome and is involved in the development of multiple organ systems, including the heart. We have examined the interaction of PITX2 isoforms with myocyte-enhancing factor 2A (MEF2A), which is a known regulator of cardiac development. A direct interaction between PITX2a and MEF2A was demonstrated using yeast two-hybrid and GST pull-down assays. To study the functional significance of this interaction, we used the atrial natriuretic factor (ANF) promoter. Coexpression of MEF2A and PITX2a or Pitx2c resulted in a strong synergistic activation of the ANF promoter in LS8 oral epithelial cells but not in other cell lines (NIH/3T3, Chinese hamster ovary, or C2C12). The synergism was dependent on promoter context, because it required MEF2 binding sites and was not seen with two other PITX2 target promoters. DNA binding by MEF2A was required but not sufficient for synergism. Upstream activators of p38 MAP kinases, MKK3 and MKK6, increased PITX2a and Pitx2c activity to yield up to 90-fold activation of the ANF promoter in LS8 cells. Because Axenfeld-Rieger syndrome is autosomal dominant and affects development of the oral epithelium, we tested one of the known PITX2 mutants. The PITX2a-K88E mutant protein suppressed wild type PITX2a synergism with MEF2A. These results demonstrate a promoter- and cell-specific functional interaction between PITX2 and MEF2A and suggest the possibility of coordinate control by these factors in the oral epithelium.
Genetics Program, University of Iowa, Iowa City, Iowa 52242, USA.
Rafael
R
Toro
Irfan
I
Saadi
Adisa
A
Kuburas
Mona
M
Nemer
Andrew F
AF
Russo
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; D051379:Mice; D032446:Myoblasts / Q000378:metabolism; D018006:Myogenic Regulatory Factors; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000097577:Homeobox Protein PITX2
D009320:Atrial Natriuretic Factor; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D048051:p38 Mitogen-Activated Protein Kinases; D000097577:Homeobox Protein PITX2; C578035:MEF2A protein, human; C578036:Mef2a protein, mouse