Molecular Mechanisms of Cardiomyocyte Differentiation

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In search for proteins that interact with the cardiac homeobox transcription factor Csx/Nkx2-5, we identified a novel protein Cal (CSX-associated LIM protein) containing LIM domains and Zim1 containing Kruppel-associated box (KRAB) and 11 zinc fingers by using a yeast two-hybrid screening. Csx/Nkx2-5 and Cal associated with each other via the LIM domains of Cal and the homeodomain of Csx/Nkx2-5. Cal itself possessed the transcription-promoting activity, and co-transfection of Cal enhanced Csx/Nkx2-5-induced promoter activation. Cal contained a functional nuclear export signal and shuttled from the cytoplasm into the nucleus in response to calcium. Accumulation of Cal in the nucleus of P19CL6 cells promoted cardiomyocyte differentiation. Zim1 was localized at the nucleus of cardiomyocytes and was associated with N-terminal and homeodomain of Csx/Nkx-2.5 via its zinc fingers. In contrast to Cal, Zim1 suppressed Csx/Nkx-2.5-mediated transcriptional activation, and transient overexpression … More of Zim1 suppressed cardiomyocyte differentiation in P19CL6 cells. These results suggest that transcriptional activity of Csx/Nkx2-5 is regulated by fine-tuned combinatorial interactions with coacitvators such as Cal and corepressors such as Zim1. Furthermore, by differential display technique, we identified a novel protein kinase Midori (myocytic induction/differentiation originator), which is expressed early during cardiogenesis and is involved in cardiomyocyte differentiation of P19CL6 cells. To investigate the developmental function of Midori, we are generating Midori-deficient mice by targeted mutagenesis. A complete null allele of Midori was generated by replacing its first coding exon with a nuclear localization signal-LacZ -pA cassetete followed by a loxP-flanked pgk-neo-pA cassette. Independent 2 lines of ES cells carrying the mutated allele were injected into blastocysts from C57BL/6J mice and the male chimeras were crossed to C57BL/6J mice. We will confirm germ-line transmission, and hererozygous intercross will give rise to offsprings including Midori-deficient mice. Less