Abstract 475: Inhibition of Cardiac Myocyte Apoptosis by Zac1, an Essential Transcription Factor for Cardiac Morphogenesis
Although there are many essential cardiac transcription factors, the mechanisms of heart development and homeostasis remain poorly understood. The transcription factors have the central roles of gene expression and organ morphogenesis. To isolate a novel cardiac transcription factor, we performed gene chip analysis using cardiac myocyte differentiating embryonic stem cells and isolated Zac1, a zinc finger protein which was identified as an anti-proliferative transcription factor. This study was designed to investigate the molecular and functional characterization of Zac1 as a cardiac transcription factor.
[Methods and Result] (1) Whole-mount in situ hybridization and immunostaining were performed at mouse embryo stages E7.5, E8.5 and E9.5. Zac1 was strongly expressed in the whole heart from E8.5 embryo to adult. (2) Luciferase constructs under the control of either ANF, BNP or αMHC promoter were co-transfected with Zac-1 expression vector, and its transcriptional activity was compared with other cardiac transcription factors. Zac1 had strong transcription activity for these promoters compared with Nkx2.5, Gata4, SRF, Tbx5 and MEF2C. (3) Gel shift assay revealed that Zac1 was directly associated with ANF promoter. (4) A total of 18 kinds of Zac1 deletion mutants was constructed. Structure-function analysis revealed that the C-terminal of Zac1 had transcriptional activity and the N-terminal had DNA binding activity. (5) Zac1 DNA binding site within the ANF promoter was also determined. It was adjacent to the Nkx2.5 binding site. Luciferase analysis revealed that Zac1 has a synergistic transcriptional activity with Nkx2.5. GST pull down analysis showed that Zac1 5th and 6th zinc finger domains directly bound to Nkx2.5 homeodomain. (6) Zac1 knock-out mouse showed severe cardiac deformity at the embryonic stage. (7) Zac1 knock-out mice showed decreased levels of several cardiac genes. (8) Genechip analysis revealed that the absence of Zac1 expression resulted in the loss of the apoptosis inhibitor gene Ifi202, thereby increasing the number of apoptotic cells in the heart.
[Conclusions] These findings indicated that Zac1 is one of the essential cardiac transcription factors, and it inhibits cardiac apoptosis.