Fig 2: Altered histone modifications and Pol II interactions at the ?-globin promoter regions and at the HBG-4kb HS in K562 cells harboring the HBG-4kb ZF. K562 cells (Untreated) and K562 cells exposed to the HBG-4kb ZF were subjected to ChIP using antibodies specific for Pol II, H3K4me3, H3K4me1, H3K27ac, or IgG. The precipitated DNA was analyzed using primers specific for the HBD-1kb HS, the HBBP1 region (Fig. 1A), the A?-globin promoter (HBG1 promoter), the G?-globin promoter (HBG2 promoter), the HBG-2kb site, the HBG-3kb site, the HBG-4kb HS, the HBG-8kb site, and the HBE1.5kb site. The error bars reflect the SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 3: Reduced ?-globin gene expression in PBMC-derived human erythroblasts after delivery of the HBG-4kb ZF. (A) Histone modifications at the G?-globin upstream region in human fetal liver HPSCs before (day 0) and after (day 5) induction of differentiation (20). (B) Western blotting of the HBG-4kb ZF (ZF-DBD), GAPDH, ?-globin, and ß-globin proteins in untreated differentiating PBMCs, in PBMCs treated with buffer, and in PBMCs treated with 1 µM HBG-4kb ZF for 24 h or with 2 µM HBG-4kb ZF for 48 h, with the ZF-containing medium being changed after 24 h. (C) Expression analysis of genes in erythroblasts in the absence or presence of the HBG-4kb ZF or the negative-control ZF-DBD (Control ZF). The ZF proteins were delivered to PBMC-derived erythroblasts for 12 h. RNA was extracted from erythroblasts incubated with the HBG-4kb ZF (top), with the control ZF (bottom), or without ZF-DBD (Untreated) and reverse transcribed. The cDNA was analyzed by qPCR using primers specific for the ?-globin (HBG), G?-globin (HBG2), adult ß-globin (HBB), embryonic e-globin (HBE), and GATA1 genes. Error bars reflect the SEM from three independent experiments (***, P < 0.001; *, P < 0.05).

Fig 4: 3'HS1 modulates the hemoglobin gene expression in ß-globin gene cluster.(A) Genome-wide Hi-C interaction map and regulatory landscape around ß-globin gene cluster in human HUDEP2 cells. ATAC-seq and CTCF track of HUDEP2 cells (Liu et al., 2018) is shown in the lower panel. Black cycle indicates the position of loops previously identified (Huang et al., 2017). Yellow dotted line indicates the three sub-TAD domains identified previously (Huang et al., 2017). HPFH1-7 deletion is illustrated and 3'HS1 is marked in blue shade. (B). The scheme of CTCF binding motif orientation engineering in HUDEP-2 cells. (C–E) In situ Hi-C contact map around ß-globin gene cluster in HUDEP-2 cells of wild type (C), 3'HS1 deletion (D), and 3'HS1 inversion (E). CTCF CUT&RUN tracks of WT (Liu et al., 2018), 3'HS1 deletion and 3'HS1 inversion HUDEP-2 cells are shown on the top of corresponding Hi-C plots. All loops that called in the HUDEP2 cells of three genotypes are marked with circles of different colors. (F) The HiCCUPS quantification of loops interaction strength by q value in ß-globin locus. Dotted line annotates q = 0.1. n.d.: not detected by HiCCUPS (q value > 0.1). (G) The composition of ß-like globin HUDEP-2 cells with 3'HS1 deletion. qPCR measurement of ß-like globin HUDEP-2 in two clones (B6 and D3) of ?3'HS1 HUDEP-2 cells is shown. Mean ± SD is displayed, n = 3. (H) Left panel: relative expression of HBE, HBG (probe measures both HBG1 and HBG2), and HBB in the 3'HS1 deleted HUDEP-2 clone B6. Mean ± SD is displayed, n = 3. Right panel: relative expression of HBE, HBG (probe measures both HBG1 and HBG2), and HBB in the 3'HS1 inverted HUDEP-2 clone A2. Mean ± SD is displayed, n = 3. (I) The right panel shows the High-performance liquid chromatography (HPLC) for globin composition in Cas9-treated HUDEP-2 control and 3'HS1 deletion clone B6. (J) Flow cytometry plot of HbF in HUDEP-2 cell clones with 3'HS1 deletion (B6 and D3), 3'HS1 inversion (A2 and G3), and ?HS5 clone.

Fig 5: Reduced ?-globin expression in K562 cells exposed to the HBG-4kb ZF. (A) Temporal effect of the HBG-4kb ZF on gene expression in K562 cells. K562 cells were exposed to 500 nM HBG-4kb ZF for 6 to 24 h, as indicated. Untreated cells were also examined as indicated. Cells were lysed, the lysates were compartmentalized into nuclear and cytoplasmic fractions, and the proteins were subjected to Western blotting using antibodies specific for CTCF, GAPDH, HBG-4kb ZF (ZF-DBD), ?-globin, Brg1, and GATA1, as shown. (B) Effect of the HBG-4kb ZF on ?-globin and GATA1 RNA and protein levels during the course of exposure to the HBG-4kb ZF. Proteins and RNA were extracted and subjected to Western blotting (using ?-globin- and GATA1-specific antibodies) or RT-qPCR using primers specific for the ?-globin (HBG) or GATA1 genes. The RT-qPCR data were from three independent experiments, and the error bars represent the SEM. (C) Assessment of ZF-DBD dosage effect. K562 cells were exposed to different concentrations (5, 50, and 500 nM) of the HBG-4kb ZF for 24 or 48 h. After exposure to the ZF-DBD, the K562 cells were subjected to Western blotting experiments with antibodies specific for GAPDH, ?-globin, or the HBG-4kb ZF (aZF). The graph on the bottom reflects the quantitation of signals from 3 independent experiments, with the error bars representing the SEM. (D) Exposure of K562 cells to the HBG-4kb ZF specifically reduced ?-globin gene expression. K562 cells were exposed to 500 nM HBG-4kb ZF (top) or to 500 nM negative-control ZF-DBD (bottom) for 12 h. RNA was isolated, reverse transcribed, and subjected to qPCR using primers specific for the ?-globin (HBGs), G?-globin (HBG2), adult ß-globin (HBB), embryonic e-globin (HBE), and GATA1 genes, as indicated. Expression in K562 cells not exposed to the ZF-DBDs (Untreated) was set at 1. Error bars reflect the SEMs from three independent biological repeats (**, P < 0.01).