We have recently identified and cloned a novel homeobox gene expressed throughout cardiac development that we have called Toto. Toto is an unusual homeobox gene for two reasons. First, it encodes a small protein (73 amino acids) that is composed almost entirely of a homoedomain. Second, it does not contain certain amino acid residues conserved amongst all other homeodomains that contact DNA, and Toto does not bind DNA. Toto encodes an 8Kd nuclear protein and it is expressed shortly after cardiac myocyte determination following expression of Nkx2.5. Toto is markedly down-regulated in Nkx2.5 null embryos, and Nkx2.5 can directly activate the Toto promoter in vitro. We have inactivated Toto in the mouse using a strategy that results in the expression of LacZ in the Toto expression domain. A significant proportion of Toto null embryos die during mid gestation with pericardial effusions and a thinned compact layer of the myocardium, sometimes associated with cardiac rupture and pericardial hematoma. Some Toto null mice live to adulthood. Preliminary data suggests that these mice have abnormal hearts. In cultured cells, Toto negatively regulates cardiac specific transcriptional pathways including those that involve Nkx2.5, Gata4, SRF and myocardin. We hypothesize that Toto functions by directly interacting with cardiac specific transcription factors to negatively regulate SRF-dependent cardiac transcription. Thus, Toto represents a new class of homeodomain proteins that has retained protein-protein interaction capabilities, but has lost sequence specific DNA binding capacity. We will clarify Toto function by addressing the following aims: 1. We will determine whether Toto inhibits Nkx2.5, Gata4 and myocardin activation of SRF-dependent gene trancription by disrupting the association of myocardin, Nkx2.5 and/or Gata4 with SRF, by interacting directly with myocardin, Nkx2.5, Gata4 and/or SRF, or by preventing SRF from interacting with DNA. 2. We will over-express Toto in the developing heart of transgenic mice to determine if cardiac-specific gene transcription is down-regulated by Toto over-expression in vivo. 3. We will determine the role of Toto deficiency in adult cardiac function under normal conditions and after hypertrophic stimuli.
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