Fig. 4.

Interference of the gene regulatory network of HNF1B inhibited PT differentiation. (A) Coverage plots showing ATAC peaks and gene expression level of HNF1B gene of the merged kidney organoid differentiation multiome dataset. Green box indicates promoter region; pink boxes indicate putative enhancer regions. (B) Corresponding integrative genomics viewer (IGV) images showing target sites of H3K4me3 (promoter, Upper), H3K27Ac (enhancer and promoter, Lower), and IgG (negative control) antibodies in CUT&RUN sequencing with day 26 organoid-derived proximal tubule (ORG_PT) and human adult kidney (HAK). (C) Schematic of HNF1B CRISPRi experiment. (D and E) qRT-PCR results of human primary RPTEC with CRISPRi for targeting promoter (D) and putative enhancer (E) regions of HNF1B. n = 3 biological replicates. The data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. (F) qRT-PCR results of kidney organoid differentiation time course with CRISPRi targeting promoter and enhancer_2 regions of HNF1B. Control samples were treated with a dCAS9-KRAB vector without sgRNA. n = 4 biological replicates. The data are presented as mean ± SEM. (G) Immunofluorescence images of HNF1B (green), LTL (white), CAS9 (red), and nuclear DAPI (blue) staining in kidney organoids (day 26) with CRISPRi for HNF1B using control vector (dCAS9-KRAB without sgRNA, Left), sgRNA targeting promoter (Middle), and sgRNA targeting enhancer_2 (Right). (Scale bars indicate 50 µm). (H) Flow cytometry analyses showing LTL-positive cell proportions of kidney organoid (day 26) with CRISPRi for HNF1B using control vector (dCAS9-KRAB without sgRNA, Left), sgRNA targeting promoter (Middle), and sgRNA targeting enhancer_2 (Right). n = 4 biological replicates. The data are presented as mean ± SEM.