Figure 5.
Linear recruitment enhancer reactivates embryonic genes in HUDEP-2 cells. (A) Schematic overview of the HBB locus on chromosome 11. The scissors indicate the binding site of the promoter that targeted gRNA (brown) with 125 bp distance to TSS and at HS1 or HS2 (light blue). (B) RT-qPCR analysis of HBE (brown), HBG1 (green), and HBG2 (pink) of transduced, bulk-proliferating cells. The mRNA levels were normalized to actin mRNA and were displayed as a percentage of HBB + HBE + HBG1/2. (C) HbE of bulk HBE125-HS1 or HS2 as measured by HPLC 3 days after differentiation. We calculated the percentage of HbF over the total Hb tetramers. (D) RT-qPCR analysis of HBE for untransduced (UT) and 5 average heterozygous HBE125-HS1 deletion clones. The mRNA levels were normalized to actin mRNA and displayed as a percentage (± SEM) of HBB + HBE + HBG1/2. (E) Schematic overview of the HBA locus on chromosome 16 depicting the R2 enhancer (red), HBZ (blue), HBA1/2 (orange), and NPRL3 (lime green) genes. The red scissors displays the gRNA at the R2 enhancer. Below that is a zoom-in on HBZ showing the binding sites of the 4 gRNAs (blue scissors) with the distance to the HBZ TSS. (F) RT-qPCR analysis of the HBZ mRNA levels in proliferating (day 0) bulk nucleofected HUDEP-2 cells that recruited R2 to different distances from the HBZ TSS. The HBZ mRNA expression was normalized to actin mRNA and is displayed as a percentage HBZ of HBA plus HBZ. (G) A schematic representation of deletions performed in the HBA locus. Two gRNAs at the HBZ promoter were either combined with a gRNA at the R2 enhancer or 490 bp upstream of the NPRL3 TSS. The dashed lines represent deletions obtained upon treatment with the 2 indicated gRNAs. (H) Same as panel F, but the RT-qPCR was performed in heterozygous-deleted HUDEP-2 lentivirus-transduced clonal cell lines. Average of 2× WT cell lines derived in the same experiment and clonal outgrowth that was genetically unrearranged. The percentages displayed as averages ± SEM of 2 to 3 clones. (I) Same as panel H but for clones differentiated for 3 days. (J) RT-qPCR results of R2-HBZ deletion clones, normalized to the HBZ levels in proliferating cells (± SEM). (K) Same as panel J but in differentiated cells, normalized to proliferating conditions.

Linear recruitment enhancer reactivates embryonic genes in HUDEP-2 cells. (A) Schematic overview of the HBB locus on chromosome 11. The scissors indicate the binding site of the promoter that targeted gRNA (brown) with 125 bp distance to TSS and at HS1 or HS2 (light blue). (B) RT-qPCR analysis of HBE (brown), HBG1 (green), and HBG2 (pink) of transduced, bulk-proliferating cells. The mRNA levels were normalized to actin mRNA and were displayed as a percentage of HBB + HBE + HBG1/2. (C) HbE of bulk HBE125-HS1 or HS2 as measured by HPLC 3 days after differentiation. We calculated the percentage of HbF over the total Hb tetramers. (D) RT-qPCR analysis of HBE for untransduced (UT) and 5 average heterozygous HBE125-HS1 deletion clones. The mRNA levels were normalized to actin mRNA and displayed as a percentage (± SEM) of HBB + HBE + HBG1/2. (E) Schematic overview of the HBA locus on chromosome 16 depicting the R2 enhancer (red), HBZ (blue), HBA1/2 (orange), and NPRL3 (lime green) genes. The red scissors displays the gRNA at the R2 enhancer. Below that is a zoom-in on HBZ showing the binding sites of the 4 gRNAs (blue scissors) with the distance to the HBZ TSS. (F) RT-qPCR analysis of the HBZ mRNA levels in proliferating (day 0) bulk nucleofected HUDEP-2 cells that recruited R2 to different distances from the HBZ TSS. The HBZ mRNA expression was normalized to actin mRNA and is displayed as a percentage HBZ of HBA plus HBZ. (G) A schematic representation of deletions performed in the HBA locus. Two gRNAs at the HBZ promoter were either combined with a gRNA at the R2 enhancer or 490 bp upstream of the NPRL3 TSS. The dashed lines represent deletions obtained upon treatment with the 2 indicated gRNAs. (H) Same as panel F, but the RT-qPCR was performed in heterozygous-deleted HUDEP-2 lentivirus-transduced clonal cell lines. Average of 2× WT cell lines derived in the same experiment and clonal outgrowth that was genetically unrearranged. The percentages displayed as averages ± SEM of 2 to 3 clones. (I) Same as panel H but for clones differentiated for 3 days. (J) RT-qPCR results of R2-HBZ deletion clones, normalized to the HBZ levels in proliferating cells (± SEM). (K) Same as panel J but in differentiated cells, normalized to proliferating conditions.

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