Fig 1.
Characterization of pathogenic missense variants in BCL11A.
(A) Left panel: Schematic of pathogenic de novo deletion, nonsense/frameshift, and missense mutations impacting BCL11A. Right panel: Fetal hemoglobin levels of the three categories of variants compared with control cohort. (B) Schematic representation of 4 reported BCL11A missense mutations and their locations in a C2HC zinc-finger (ZnF0) at the N-terminal region of BCL11A isoforms (BCL11A-S, BCL11A-L, BCL11A-XL) and the fifth zinc finger domain (ZnF5) at the C-terminal region of BCL11A-XL. (C) Distribution of BCL11A genetic variants from the gnomAD database.
Fig 2.
Functional assessment of missense variants impact on HbF silencing.
(A) Workflow of exogenous expression of either the wild type or mutant forms of the BCL11A XL in cord blood HSPCs showing lentiviral transduction, GFP sorting, erythroid differentiation and functional analysis. (B) Left panel: A histogram plot shows GFP levels indicating robust and equivalent expression (GFP linked to exogenous BCL11A-XL cDNA). Right panel: A representative western blot and the normalized quantification of BCL11A levels from exogenous cDNA expression in cord blood erythroid cells. BCL11A relative expression levels are normalized with GAPDH expression. Band intensities of all groups are relative to BCL11A-XL WT group. Results are shown as mean ± SEM from three biological replicates. (C) Gene expression analysis for HBG1/2 and HBB in cord blood erythroid cells expressing exogenous isoforms of BCL11A cDNA. Results are shown as mean ± SEM from three biological replicates. (D) Representative HbF flow cytometry on day 13 of cord blood erythroid differentiation. Results are shown as mean ± SEM from three biological replicates. (E) Representative HbF HPLC on day 17 of cord blood erythroid differentiation. HbF: hemoglobin F (fetal form); HbA: hemoglobin A (adult form). HbF%: HbF as a fraction of HbA plus HbF.
Fig 3.
Examining protein stability and cellular thermal shifts of BCL11A missense variants.
(A) Workflow of exogenous expression of either the wild type or mutant forms of the BCL11A XL in K562 cells showing lentiviral transduction, GFP sorting, protein stability and cellular thermal shifts assays. (B) Quantitative representation of BCL11A levels for Cycloheximide Chase Assay from exogenous wild type or mutant forms of the BCL11A XL cDNA expression in K562 cells. Cells were treated with 25 μg/ml cycloheximide to arrest ongoing protein synthesis and were collected for western blotting of BCL11A expression at 0hr, 6hr, 12hr and 24hr time points post cycloheximide treatment. BCL11A relative expression levels are normalized with GAPDH expression. Results are shown as mean ± SEM from three biological replicates. (C) Quantitative representation of western blotting of BCL11A levels for MG-132 treatment (10 μM, 4hr) from exogenous wild type or mutant forms of the BCL11A XL cDNA expression in K562 cells. BCL11A relative expression levels are normalized with GAPDH expression. Band intensities of all groups are relative to 0hr time point of each group. Results are shown as mean ± SEM from three biological replicates. (D) Protein disorder prediction and comparison for BCL11A mutant forms relative to wild type form using IUPred disorder score. (E) Quantitative modeling of BCL11A levels for Cellular Thermal Shift Assay from exogenous wild type or mutant forms of the BCL11A XL cDNA expression in K562 cells. Cells were treated at 27°C, 37°C, 47°C, 57°C, and 67°C for 3 minutes and were collected for western blotting of BCL11A expression. BCL11A relative expression levels are normalized with GAPDH expression. Results are shown as mean ± SEM from three biological replicates. (F) Melting temperatures (Tm) for wild type or mutant forms of the BCL11A based on modeling of BCL11A levels for Cellular Thermal Shift Assay. Results are shown as mean ± SEM from three biological replicates.
Fig 4.
Loss of DNA binding by a pathogenic missense variant in BCL11A ZnF5 underlies impaired HbF silencing.
(A) Left panel: Structure of ZnF5 for wild type and Mut 4 (p.Lys784Thr) form of BCL11A. Right panel: Zoom in view of ZnF5 indicating DNA binding alteration between wild type (Lys-784) and Mut 4 (Thr-784). (B) Workflow of exogenous expression of either the wild type or Mut 4 forms of the BCL11A XL with a HA-tag at C terminal in cord blood HSPCs showing lentiviral transduction, GFP sorting, erythroid differentiation, functional and binding analysis. (C) Western blot of BCL11A expression for nuclear/cytoplasmic fractions using antibody against HA-tag in cord blood erythroid cells expressing exogenous wild type and Mut 4 HA-tagged forms of BCL11A. LMNB1 and GAPDH as controls for nuclear fraction and cytoplasmic fraction. Representative results are shown from three biological replicates. (D) Gene expression analysis for HBG1/2 and HBB in cord blood erythroid cells expressing exogenous wild type and Mut 4 HA-tagged forms of BCL11A. Results are shown as mean ± SEM from three biological replicates (***P<0.001, n.s.: statistically non-significant by a two-tailed Student t-test). (E) Representative HbF flow cytometry on day 13 of cord blood erythroid differentiation. Results are shown as mean ± SEM from three biological replicates. (F) Chromatin binding activities of BCL11A across within the human β-globin locus in cord blood-derived erythroid cells expressing the wild type or Mut 4 HA-tagged proteins at day 11 of erythroid differentiation. Results are shown as mean ± SEM from three biological replicates (***P<0.001 by a two-tailed Student t-test).