Abstract 691: Zac1 Is An Essential Cardiac Transcription Factor
Although there are many essential cardiac transcription factors, the mechanisms of heart development and homeostasis remain poorly understood. The transcription factors have the central role of gene expression, pathogenesis and organ morphogenesis. To isolate the novel cardiac transcription factor, we performed gene chip analysis by using cardiac myocyte differentiating embryonic stem cells and isolated the 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]
Whole-mount in situ hybridization and immunostaining of Zac1 was 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.
Luciferase constructs under the control of either ANF, BNP and 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.
Gel mobility shift assay revealed that Zac1 was directly associated DNA within ANF promoter.
The 18 kinds of Zac1 deletion mutants were constructed. Structure-functional analysis revealed that the C-terminal of Zac1 had transcriptional activity and N-teminal had DNA binding activity.
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.
Zac1 has a PKC phosphorylation site. PKC activation increased the wild type Zac1 transcriptional activity. Zac1 T167N was mutated at the PKC phosphhorylation site. Zac1 T167N abolished PKC-dependent transcriptional activity.
Zac1 knock-out mouse showed severe cardiac deformity at the embryonic stage.
[Conclusions] These findings indicated that Zac1 is one of the essential cardiac transcription factors, and it collaborates with other cardiac transcription factors.