Integrated single cell RNA-seq and ATAC-seq of iPSC-derived kidney organoids reveals the complexity of TGF beta signalling to chromatin during the origin of fibroblasts

Differential methylation patterns and changes in post-translational histone modifications are observed in patients with or without progressive diabetic kidney disease (DKD). TGFβ1 plays a central regulatory role in the determination of renal cell fate and the progression of DKD. Using ChIP-seq, we identified an association between SMAD3 and the histone methyltransferase, EZH2, in iPSCs and in iPSC derived nephron progenitors. Using the 10X Genomics platform, we performed single cell RNA-seq and -ATAC-seq on human iPSC-derived kidney organoids treated with the EZH2 specific inhibitor, GSK343, prior to treatment with TGFβ1. Integration of single cell RNAseq and -ATACseq identified de novo ACTA2+ve/POSTN+ve myofibroblasts in TGFβ1-treated kidney organoids, characterised by increased SMAD3-dependent cis chromatin accessibility and the expression of genes associated with fibroblast activation, which are similarly overexpressed in DKD patients. This fibrosis-associated regulon is characterised by enrichment of SMAD3 and AP-1, corresponding to FOS/JUN motifs at accessible loci identified by scATACseq. Treatment with GSK343 blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. We propose that that the enzymatic function of the polycomb repressive complex is necessary for TGFβ1 induced increase in chromatin accessibility and its subsequent gene regulatory functions. Manipulation of the association between SMAD3 and EZH2 may be a useful therapeutic strategy for the resolution of renal fibrosis.