Extended Data Fig. 5: ZNF410 represses HbF by activating CHD4.
From: ZNF410 represses fetal globin by singular control of CHD4
a, Comparison of genes downregulated in ZNF410 and CHD4 mutant cells by GSEA (b) LC3-I/II and GAPDH (control) immunoblot in unedited parental and ZNF410 null HUDEP-2 cells (left panel) and mock and ZNF410 targeted primary erythroblasts (c) Correlation of ZNF410 and CHD4 expression across 54 human tissues from GTEx (Pearson r = 0.77, p < 0.0001) (d) CHD4 expression in ZNF410 targeted (n = 3) compared to mock (n = 1) and AAVS1 (n = 1) targeted control HUDEP-2 cells. Data are mean values, error bars are standard deviation. e, Cas9 paired cleavages with CHD4-proximal-gRNA-1 and CHD4-distal-gRNA-1 (CHD4 Δ 6.7 kb) or CHD4-proximal-gRNA-1 and CHD4-distal-gRNA-2 (CHD4 Δ 6.9 kb) were used to generate HUDEP-2 clones with biallelic deletions spanning both of the ZNF410 binding regions upstream of CHD4. Positions of ZNF410 motifs (red rectangles) and accessible chromatin by ATAC-seq (gray peaks) (f) CHD4 expression in CHD4 Δ 6.9 kb clones (n = 3) compared to HUDEP-2 cells (n = 1) (left panel) and HbF level measured by hemoglobin HPLC in CHD4 Δ 6.7 kb (n = 1) and Δ 6.9 kb (#2 and #3, n = 2) clones compared to HUDEP-2 cells (n = 1) (right panel). g, CHD4 Δ 6.9 kb clones and HUDEP-2 cells were subjected to AAVS1 (negative control), ZNF410 and ZBTB7A targeting using RNP electroporation of 3X-NLS-Cas9 and sgRNA. Left panel, editing efficiency measured by indel frequency in HUDEP-2 cells (n = 1) and CHD4 Δ 6.9 kb clones (n = 3) targeted with ZNF410 or ZBTB7A sgRNAs. The shaded portion of the bar represents the percentage of indels resulting in frameshift (fs) alleles. The white portion of the bar represents in-frame indels. Right panel, HBG expression relative to total β-like globin (HBG + HBB) in HUDEP-2 cells (n = 1) and CHD4 Δ 6.9 kb clones (n = 3) targeted with AAVS1 (negative control), ZNF410 or ZBTB7A sgRNAs. h, HBG expression relative to total β-like globin (HBG + HBB) in CHD4 Δ 6.9 kb clone 3 (n = 1) subjected to ZNF410, BCL11A and ZBTB7A targeting using RNP electroporation of 3xNLS-Cas9 and sgRNA compared to mock (n = 1) cells. i, CBX6 expression in mock, AAVS1 and ZNF410 targeted HUDEP-2 cells (n = 2 for mock and control, n = 3 for ZNF410 targeted) and CHD4 Δ 6.7 kb HUDEP-2 cells (n = 3 for mock, control and ZNF410 targeted). Catalase was used as the endogenous normalization control. CBX6 expression in targeted cells is shown relative to expression in mock cells. Data are mean values, error bars are standard deviation.
