Abstract OEC101: Cardiac Reprogramming Inducer ZNF281 Is Indispensable For Heart Development By Interacting With Key Cardiac Transcriptional Factors

Abstract

During direct cardiac reprogramming, cardiogenic transcription factors (TFs) cooperatively reshape the epigenomic landscape by activating cardiac enhancers, which describe a common epigenetic signature between in vitro cardiac reprogramming and in vivo heart development. We thus hypothesized that direct reprogramming could be a useful tool to study novel transcriptional regulators in heart development. To test this hypothesis, we focused on ZNF281/Zfp281(Zinc finger protein 281), a pluripotency regulator which showed up as an unexpected strong cardiac reprogramming inducer from our previous unbiased screen. Since Zfp281 is an essential factor for epiblast maturation, we established a Zfp281 floxed mouse line using CRISPR/Cas9 system to study the impact of Zfp281 after cardiac lineage commitment. We then generated multiple conditional Zfp281KO (cKO) mouse lines by crossing with different stage-specific Cre driver lines. Surprisingly, we found that mesodermal (Mesp1-Cre) Zfp281cKO mice were embryonic lethal as early as E9.5, associated with various cardiac anomalies. In contrast, cardiac progenitor (Nkx2-5-Cre) and cardiomyocyte (Myh6-Cre) Zfp281cKO mice survived to adulthood and appeared grossly normal, suggesting a crucial role of Zfp281 in mesodermal to cardiac differentiation. Next, we performed RNA-seq analysis to study the molecular mechanism of Zfp281 deficient cardiac anomalies. Interestingly, expression of cardiac TFs and sarcomeric genes were altered in Zfp281cKO embryos compared to control siblings. Consistently, co-immunoprecipitation analysis showed direct interaction of Zfp281 with Mesp1 and Gata4. These data indicate that Zfp281 directly interacts with cardiac TFs to cooperatively orchestrate the cardiogenic transcriptional network. In conclusion, by using multiple genetic mouse models, we discovered that cardiac reprogramming inducer Zfp281 plays a pivotal role in heart development. Although GWAS studies have identified various disease-causing mutations, the etiology for the majority of congenital heart disease remains largely unknown and there are still such missing pieces to be uncovered in the interdependent cardiogenic transcriptional network. Our study demonstrates and highlights the impact and possibility of direct reprogramming as a novel approach to study cardiovascular biology.