Journal: Nature genetics

Article Title: Transcription factor competition at the γ-globin promoters controls hemoglobin switching

doi: 10.1038/s41588-021-00798-y

Figure Lengend Snippet: (a) Flow chart of the dense perturbation experiment design. (b) Zoomed in view of the dense perturbation results at HBB and HBD genes. gRNAs that target the exons are enriched in Cas9 experiment. HbF raw score is enrichment of individual gRNAs in HbF-high compared to unsorted population at end of erythroid maturation, plotted as log 2 fold change. HbF score shows deconvoluted underlying genomic regulatory signal with corresponding p-values shown on −log 10 scale. (c) Zoomed in view of the dense perturbation results at HBG1 gene. Not that gRNAs that target the exons are depleted in Cas9 experiment. (d) Zoomed in view of the dCas9 dense perturbation result at HS3 of the LCR aligned to PhastCons46way scores. The four regions highlighted in green contain GATA1 or GATA1-TAL1 composite motifs (CTG[N8–9]GATA), with the sequences shown below. (e) RT-qPCR showing that dCas9/sgRNA binding at −115 of γ-globin promoters reduced γ-globin expression in HUDEP-2 cells. Note that the γ-globin is only expressed at a basal level in cells expressing AAVS1 control sgRNA. The result is shown as mean (SD) of three technical replicates. Statistical tests of the beta coefficients were performed empirically through bootstrapping and two-tailed tests. Multiple hypothesis testing was accounted for with the Benjamini-Hochberg (BH) procedure.

Article Snippet: HUDEP-2 cells were transduced with lentivirus encoding various Cas9 or dCas9 variants (Cas9: LentiCas9-Blast, Addgene plasmid #52962 ; dCas9: pGH125_dCas9-Blast, Addgene plasmid #85417 ; dCas9-KRAB: pHR-SFFV-dCas9-BFP-KRAB, Addgene plasmid #46911 ; dCas9-VP64: pHRdSV40-dCas9–10xGCN4_v4-P2A-BFP, Addgene plasmid #60903 and pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS, Addgene plasmid #60904 ) to produce stably expressing cells.

Techniques: Quantitative RT-PCR, Binding Assay, Expressing, Control, Two Tailed Test

Journal: Nature genetics

Article Title: Transcription factor competition at the γ-globin promoters controls hemoglobin switching

doi: 10.1038/s41588-021-00798-y

Figure Lengend Snippet: (a) Dense perturbation of genomic sequences around the β-globin gene cluster by pooled gRNA library in Cas9, dCas9, dCas9-KRAB or dCas9-VP64 expressing HUDEP-2 cells. gRNA enrichment in HbF-high cells was used to deconvolute underlying genomic regulatory signal (with “HbF” track displaying beta coefficient). Corresponding p-values are shown on −log 10 scale. (b) A zoomed-in view of the dense perturbation results at the γ-globin ( HBG1 ) promoter and first exon. “HbF raw” scores were calculated as enrichment of reads for each sgRNA in HbF-high compared to the unsorted population at end of erythroid maturation (shown as log 2 fold change). “HbF” track depicts the deconvoluted regulatory signal as beta coefficient with associated p-values below. Minor ticks indicate value of zero. The result at the HBG2 promoter was the same as the sequences share 99.3% identity. (c) Schematic structure of γ-globin promoters. The binding sites of LRF/ZBTB7A, BCL11A, and TBP are indicated. Sequence conservation of HBG1 and HBG2 promoters across 46 vertebrates (phastCons46way) are shown as cyan and orange lines, respectively. Two CCAAT boxes that are potential binding sites of NF-Y are highlighted in red. The distal TGACCA motif through which BCL11A represses γ-globin expression is delineated with a rectangle. Statistical tests of the beta coefficients were performed empirically through bootstrapping and two-tailed tests. Multiple hypothesis testing was accounted for with the Benjamini-Hochberg (BH) procedure.

Article Snippet: HUDEP-2 cells were transduced with lentivirus encoding various Cas9 or dCas9 variants (Cas9: LentiCas9-Blast, Addgene plasmid #52962 ; dCas9: pGH125_dCas9-Blast, Addgene plasmid #85417 ; dCas9-KRAB: pHR-SFFV-dCas9-BFP-KRAB, Addgene plasmid #46911 ; dCas9-VP64: pHRdSV40-dCas9–10xGCN4_v4-P2A-BFP, Addgene plasmid #60903 and pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS, Addgene plasmid #60904 ) to produce stably expressing cells.

Techniques: Genomic Sequencing, Expressing, Binding Assay, Sequencing, Two Tailed Test

Journal: Nature genetics

Article Title: Transcription factor competition at the γ-globin promoters controls hemoglobin switching

doi: 10.1038/s41588-021-00798-y

Figure Lengend Snippet: (a) The sequence of γ-globin promoters. The eight sgRNAs used for dCas9 disruption experiment are shown below the sequence and color coded. The numbers (−208, etc) represent the distances between TSS and positions of the 17 th nucleotide of each sgRNA. The position of LRF, BCL11A and NF-Y motifs are labeled in red. The flanking sequences of NF-Y motif is also involved in NF-Y binding. (b) Right, RT-qPCR analysis of the percentage of γ-globin in BCL11A KO HUDEP-2 cells expressing dCas9 with different sgRNAs. Data is showed as mean (SD) of three technical replicates and a representative of two biological replicates. Left, NFYA CUT&RUN in these cells. The cartoon indicates the deduced protein binding at the promoters.

Article Snippet: HUDEP-2 cells were transduced with lentivirus encoding various Cas9 or dCas9 variants (Cas9: LentiCas9-Blast, Addgene plasmid #52962 ; dCas9: pGH125_dCas9-Blast, Addgene plasmid #85417 ; dCas9-KRAB: pHR-SFFV-dCas9-BFP-KRAB, Addgene plasmid #46911 ; dCas9-VP64: pHRdSV40-dCas9–10xGCN4_v4-P2A-BFP, Addgene plasmid #60903 and pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS, Addgene plasmid #60904 ) to produce stably expressing cells.

Techniques: Sequencing, Disruption, Labeling, Binding Assay, Quantitative RT-PCR, Expressing, Protein Binding

Journal: Nature genetics

Article Title: Transcription factor competition at the γ-globin promoters controls hemoglobin switching

doi: 10.1038/s41588-021-00798-y

Figure Lengend Snippet: Competitive binding between NF-Y and BCL11A controls hemoglobin switching. In fetal stage erythroid cells, or cells with HPFH mutations or lacking BCL11A, NF-Y binds to the γ-globin promoters and activates expression. In adult stage erythroid cells, BCL11A prevents NF-Y binding and represses γ-globin in concert with NuRD. LRF/ZBTB7A independently recruits NuRD and represses γ-globin through binding to the −200 bp region of the γ-globin promoters (not illustrated in the model). dCas9 binding at the BCL11A motif is sufficient to disrupt NF-Y binding and repress γ-globin expression. When γ-globin is silenced, NF-Y may bind to β-globin and regulate its expression. Other known positive regulators of β-globin including GATA1, KLF1, LDB1, etc are not shown.

Article Snippet: HUDEP-2 cells were transduced with lentivirus encoding various Cas9 or dCas9 variants (Cas9: LentiCas9-Blast, Addgene plasmid #52962 ; dCas9: pGH125_dCas9-Blast, Addgene plasmid #85417 ; dCas9-KRAB: pHR-SFFV-dCas9-BFP-KRAB, Addgene plasmid #46911 ; dCas9-VP64: pHRdSV40-dCas9–10xGCN4_v4-P2A-BFP, Addgene plasmid #60903 and pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS, Addgene plasmid #60904 ) to produce stably expressing cells.

Techniques: Binding Assay, Expressing