Abstract
The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5−/− null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117′s interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.
Highlights
We previously identified several novel direct target genes for Nkx[2,3,4,5] in the Second Heart Field (SHF) region of mice during outflow tract (OFT) development[11]
Ccdc[117] mRNA expression was noted in dorsal pharyngeal mesoderm and ventral portions of the neural tube, indicating that Nkx[2,3,4,5] may regulate additional indirect and non-cell autonomous Ccdc[117] expression in these populations at this developmental stage. qRT-PCR assay confirmed the reduction of Ccdc[117] mRNA expression in SHF-containing pharyngeal arch of Nkx2-5−/− knockout embryos as compared to wild-type (Fig. 1G). These results were at odds with findings from our previous combinatorial mRNA expression microarray-based study, which predicted that Ccdc[117] was directly, but negatively, regulated by Nkx[2-511]. This previous study was based on a re-analysis of publicly available, earlier generation expression microarray data derived from analysis of more extensive E9.5 cardiothoracic regions of wild-type and Nkx2-5−/− mouse embryos than those used in our study, and included regions where we observed qualitatively less reduction of Ccdc[117] expression in the knockout (e.g., more anterior pharyngeal and more posterior lateral mesoderm (Fig. 1C))
To evaluate the likelihood of direct regulation of Ccdc[117] by Nkx[2,3,4,5] in vivo, we examined promoter and regulatory region occupancy by Nkx[2,3,4,5] in developing embryos using chromatin immunoprecipitation (ChIP) analysis
Summary
Knockdown of protein expression of CIA2B, the Ccdc117-interacting component of the MMS19 targeting complex, in HeLa cells resulted in parallel changes in the rate of DNA synthesis, DNA damage repair and cell cycle progression. These data support a functional interaction between Ccdc[117] and MMS19 in facilitating DNA repair, cell cycle progression, and cell proliferation They suggest a novel mechanistic hypothesis to explain the cardiac proliferation defects observed in Nkx[2,3,4,5] knockout whereby loss of Ccdc[117] expression in SHF progenitors leads to cell cycle delay, loss of proliferation, and OFT/right heart morphogenic defects (Fig. 8). The identification of Ccdc[117] as both a direct downstream target of Nkx[2,3,4,5] regulation, and as a functional interacting partner of the MMS19 CIA targeting complex provides a novel and insightful link between metabolic state and the cardiac morphogenic pathways underlying CHD via the harnessing of FeS synthesis, assembly, and targeting pathways regulating cell proliferation
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