MOLECULAR-CLONING AND CHARACTERIZATION OF HUMAN CARDIAC HOMEOBOX GENECSX1

Accumulating evidence has suggested that homeodomain-containing protei ns play critical roles in regulating the tissue-specific gene expressi on essential for tissue differentiation and in determining the tempora l and spatial patterns of development. In order to elucidate the mecha nisms of human heart development. we have isolated a human homologue o f the murine cardiac homeobox gene Csx (also called Nkx-2.5) and denot ed it as CSX1. The amino acid sequence of the CSX1 homeodomain is 100% and 67% identical to that of murine Csx/Nkx-2.5 and Drosophila tinman , respectively. CSX1 has at least three isoforms generated by an alter native splicing mechanism. One of these isoforms (CSX1a) encodes a pro tein of approximate to 35 kD that possesses the homeodomain, whereas t he other two (CSX1b and CSX1c) encode a truncated protein of approxima te to 12 kD that is identical to the CSX1a protein at the amino-termin al 112 amino acids but lacks the homeodomain. Northern blot analysis s howed that CSX1 transcripts are abundantly expressed in both fetal and adult hearts, but no signal was detected in other human tissues exami ned. Amplification of each isoform by reverse transcriptase-polymerase chain reaction I revealed that all of the three isoforms are expresse d in fetal and adult hearts and that the homeobox-containing isoform C SX1a is most abundant. The homeodomain-containing protein encoded by C SX1a binds to Csx/Nkx-2.5 binding sequences and transactivates the seq uence-containing luciferase reporter gene. Unexpectedly, the homeodoma in-lacking protein encoded by CSX1b also transactivates the reporter g ene, although CSX1b does not bind to the Csx/Nkx-2.5 binding sequences . The highly conserved homeodomain sequences in evolution and the rest ricted expression in the heart suggest that CSX1 plays an important ro le in the development and differentiation of the human heart and that there may be two different mechanisms in transcriptional regulation by the CSX1 protein, homeodomain-dependent and -independent mechanisms.