UCSF

GATA4 is a central transcriptional regulator during cardiac development and for postnatal function. Familial mutations in GATA4 lead to autosomal dominant congenital heart disease and cardiomyopathy. We previously reported a GATA4 G296S mutation that resides near the second zinc finger of GATA4, involved in DNA-binding and protein-protein interactions thought to be dominant due to haploinsufficiency. However, the mechanisms by which this mutation affects the transcriptional and epigenetic landscape as well as the cellular physiology is unknown. Here, we have used genomic approaches as well as cell-based assays to delineate the consequences of the GATA4 G296S mutation in cardiomyocytes.

We generated iPS cells from human patients with or without the GATA4 G296S mutation and differentiated them to a purified population of cardiomyocytes. Using these cells, we conducted investigations into the role of a putative modifier gene identified by bioinformatics analyses for its potential involvement in the development of the GATA4 cardiomyopathy. However we were not able to uncover a disease causing relationship independent of GATA4.

We performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) on endogenous GATA4 protein and revealed hundreds of previously unidentified GATA4-bound loci while confirming known GATA4 targets. We also performed ChIP-Seq on TBX5, a transcription factor associated with congenital heart disease that physically interacts with GATA4 and whose interaction is specifically disrupted by the G296S mutation. Genome-wide we identified thousands of loci where GATA4 and TBX5 co-localized in wild-type cells, but surprisingly this co-occupancy was lost in cardiomyocytes heterozygous for the GATA4 G296S mutation, despite the presence of the wildtype allele. RNA-Seq revealed dysregulation of transcription at many of these loci, along with expected epigenetic changes, with clustered dysregulation of genes involved in cellular respiration, inflammation, and muscle contraction. To investigate the effect of these genes, we undertook a number of cell-based assays. In collaboration with Beth Pruitt’s lab at Stanford, we developed a platform for performing individual cell-based force generation and contractility studies. These contractility and other cell-based studies confirmed defects in contractility and calcium handling in cardiomyocytes heterozygous for the GATA4 G296S mutation. We also verified that the heterozygous Gata4 G295S mouse has an increased susceptibility to pressure-overload induced hypertrophy and demonstrated its limited ability to validate targets dysregulated by RNA-seq.

The deep “-omic” interrogation of iPS-derived cardiomyocytes effectively revealed the transcriptional and cellular consequences of an important human mutation and may explain many of the disease features observed in individuals with GATA4 mutations. Further study is still required to delineate the full consequences of this dysregulated transcriptome, but these observations help to confirm the role of GATA4 in the heart and set a strong foundation for future investigations.