Abstract 11230: Coordinate Cistrome Regulation by Reprogramming Factors During Direct Human Cardiac Reprogramming

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Introduction: Direct cardiac reprogramming of somatic cells into induced cardiomyocytes (iCMs) through overexpressing cardiac enriched transcription factors is considered as a promising strategy for regenerative medicine and an important cell model to study cell fate determination. In addition to our published cocktail using MEF2C, GATA4, and TBX5 and microRNA miR-133 (MGT133), we found that addition of TBX20 significantly enhanced cardiac reprogramming and generated human iCMs (hiCMs) more similar to functional cardiomyocyte identity. However, it is largely unknown how the reprogramming factors coordinately regulate direct lineage conversion. Methods: To determine the regulatory networks orchestrated by transcription factors, we performed CUT&TAG or CUT&RUN for MEF2C, GATA4, TBX5, TBX20 and histone marks H3K27me3, H3K27ac and H3K4me3 in hiCMs generated with MGT133 or MGT133+TBX20. We also performed time course RNA-seq and ATAC-seq analyses to profile transcriptome and chromatin accessibility changes during reprogramming process. Results: We mapped the genome wide binding of each reprogramming factor and determined the co-occupancy of TBX20 and MGT at a majority of cardiac gene promotors, especially at genes related to cardiac cell structure and contractility, such as MYBPC3, SCN5A and MYH7. Interestingly, TBX20 itself is not able to target cardiac genes unless is expressed together with MGT. Meanwhile, expression of TBX20 led to significantly increased chromatin occupation of MGT factors and upregulation of cardiac-specific genes. Our immunoprecipitation assay further confirmed the physical interaction of TBX20 with MGT. Intriguingly, in addition to targeting cardiac-lineage genes, TBX20 also binds to many fibroblast-specific genes, such as LAMB1 and IGFN1. While binding of TBX20 augments chromatin accessibility at cardiac-specific genes loci, it decreases the chromatin openness at fibroblast genes. Conclusions: TBX20 cooperates with MEF2C, GATA4, and TBX5 to target cardiac-specific genes and facilitate gene transcription and serves as a transcription repressor of non-cardiac lineage genes. These results provided significant insight into cistrome regulation during human direct cardiac reprogramming.