ACTIVATION OF THE CARDIAC ALPHA-ACTIN PROMOTER DEPENDS UPON SERUM RESPONSE FACTOR, TINMAN HOMOLOG, NKX-2.5, AND INTACT SERUM RESPONSE ELEMENTS

A murine cardiac specific homeobox gene, Nkx-2.5/CSX, a potential Dros ophila tinman homologue, may have a fundamental role in cardiac myocyt e differentiation. DNA binding targets for Nkx-2.5 were recently shown to represent novel homeodomain binding sequences, some of which resem bled serum response elements (SREs); [Chen CY, Schwartz RJ (1995): J B iol Chem 270: 15628-15633]. In this study, Nkx-2.5 facilitated serum r esponse factor (SRF) DNA-binding activity to the multiple SREs found o n the cardiac alpha-actin promoter and together stimulated cardiac alp ha-actin promoter dependent transcription in 1OT1/2 fibroblasts. Analy sis of cardiac alpha-actin promoter mutants demonstrated the importanc e of the multiple upstream SREs and an obligatory requirement for an i ntact proximal SRE1, for providing high levels of activity in the pres ence of Nkx-2.5 and SRF coexpression. Transfection assays with mutant SRF species indicated that the C-terminal activation domain and DNA-bi nding MADS box were necessary for transcriptional activity in the pres ence of Nkx-2.5. Expression of Nkx-2.5 mutants also demonstrated that the homeodomain alone was insufficient for directing promoter activity in the presence of SRF. The central role of SRF in regulating striate d alpha-actin gene activity also was revealed by its embryonic express ion restricted primarily to myocardium of the developing heart and the myotomal portion of somites. Thus the function of the cardiac actin p romoter SREs appeared to provide binding sites for SRF and Nkx-2.5 to interact and elicit striated muscle specific transcription that was in dependent of the MyoD family. (C) 1996 Wiley-Liss, Inc.