Abstract 895: Identification of a Novel Role of ZIC3 in Regulating Cardiac Development
Mutations in ZIC3, a zinc finger transcription factor, were first identified in Heterotaxy Syndrome, a class of congenital disorders resulting from failure to establish normal left-right asymmetry. Recently, we reported ZIC3 mutations in patients with isolated congenital heart defects (CHD); however, the pathogenesis remains unknown. We hypothesized that ZIC3 regulates cardiac development independent of left-right patterning. Here, we used embryonic stem (ES) cells and knockout mouse embryos to study cardiac-specific gene expression. RT-PCR suggests that Zic3 is expressed in the ES cells, as well as the developing heart tissue. Significant reductions in atrial natriuretic factor (ANF) (>90%), Nkx2.5 (>90%), Tbx5 (>85%) were observed in Zic3null/y ES cells, while the expression levels of SRF and GATA4 remained unchanged. In situ hybridization showed that ANF is severely reduced in the developing heart of E9.5 Zic3null embryos, while Nkx2.5, Tbx5 appear normal. We observed reduced trabecular myocardium, thin ventricular walls and slightly enlarged ventricular chamber in E9.5 Zic3null embryos with no heart looping defects, similar to what was found in embryos with cardiac-specific ablation of SRF. Electrophoresis mobility shift assay and luciferase reporter assay indicated that ZIC3 binds to and inhibits the cardiac α-actin promoter through zinc finger domains. ZIC3 itself has no effect on the ANF promoter, but it functions as a coactivator of SRF on both cardiac α-actin and ANF promoters. GST pull-down assay showed that ZIC3 binds to the MADS box of SRF. Investigation of the ZIC3 mutants identified in CHD patients suggested that both N’-domain and zinc finger domains in ZIC3 are required for ZIC3-SRF physical interaction and serve as the activation domain for their functional interaction, while the C’-domain functions as an inhibitory domain. Given the fact that ANF is a direct SRF target and also a molecular marker of trabecular myocardium, we suggest that the reduced cardiac expression of ANF in Zic3null embryos results from a direct effect of ZIC3/SRF interaction on ANF promoter, as well as a secondary effect of reduced development of the trabecular myocardium. In conclusion, these findings reveal a novel role of Zic3 in regulating cardiac development.