DNA-binding affinity and specificity determine the phenotypic diversity in BCL11B-related disorders

Abstract

BCL11B is a Cys2-His2 zinc-finger (C2H2-ZnF) domain-containing, DNA-binding, transcription factor with established roles in the development of various organs and tissues, primarily the immune and nervous systems. BCL11B germline variants have been associated with a variety of developmental syndromes. However, genotype-phenotype correlations along with pathophysiologic mechanisms of selected variants mostly remain elusive. To dissect these, we performed genotype-phenotype correlations of 92 affected individuals harboring a pathogenic or likely pathogenic BCL11B variant, followed by immune phenotyping, analysis of chromatin immunoprecipitation DNA-sequencing data, dual-luciferase reporter assays, and molecular modeling. These integrative analyses enabled us to define three clinical subtypes of BCL11B-related disorders. It is likely that gene-disruptive BCL11B variants and missense variants affecting zinc-binding cysteine and histidine residues cause mild to moderate neurodevelopmental delay with increased propensity for behavioral and dental anomalies, allergies and asthma, and reduced type 2 innate lymphoid cells. Missense variants within C2H2-ZnF DNA-contacting α helices cause highly variable clinical presentations ranging from multisystem anomalies with demise in the first years of life to late-onset, hyperkinetic movement disorder with poor fine motor skills. Those not in direct DNA contact cause a milder phenotype through reduced, target-specific transcriptional activity. However, missense variants affecting C2H2-ZnFs, DNA binding, and "specificity residues" impair BCL11B transcriptional activity in a target-specific, dominant-negative manner along with aberrant regulation of alternative DNA targets, resulting in more severe and unpredictable clinical outcomes. Taken together, we suggest that the phenotypic severity and variability is largely dependent on the DNA-binding affinity and specificity of altered BCL11B proteins.

Keywords: BCL11B; C2H2-type zinc finger protein; genotype-phenotype correlation; recognition code; type 2 innate lymphoid cells.

Graphical abstract

Figure 1

Locations of identified BCL11B germline variants Schematic protein structure of BCL11B. Previously identified variants are shown in the upper panel. Variants identified in this study are shown in the lower panel. Likely gene-disruptive (LGD) variants are shown in gray. Missense variant in the very N terminus is shown in black. Missense variants affecting “specificity residues” are shown in blue, other missense variants within the α helix are shown in violet, and missense variants affecting ZnF-contacting cysteines and histidines are shown in red. Recurring variants are underlined. C, C terminus; N, N terminus; ZnF, zinc-finger C2H2 domain.

Figure 2

Location of missense variants within BCL11B’s Cys2-His2 zinc-finger domains Schematic diagrams of Cys2-His2 (C2H2-ZnF) zinc-finger domains 2–6. Each C2H2-ZnF contains paired cysteines (C) and histidines (H) that bind the zinc ion (Zn). β sheet is depicted in orange, α helix is depicted in blue, and 1, +2, +3, and +6 depict “specificity residues” within the α helix. Missense variants affecting “specificity residues” are shown in blue, other missense variants within the α helix are shown in violet, and missense variants affecting ZnF-contacting cysteines and histidines are shown in red.

Figure 3

Images of individuals affected by BCL11B-associated disorders (A) Dental photos show primary molars of individual 8 immediately after eruption (left). The other two photos were taken at the age of 9 years. The “raised” gums above the left incisor were much more pronounced after the permanent tooth erupted and have slowly but steadily grown well downward. (B) Images of individual 10 at the age of 5 years shows short forehead, thin eyebrows, “beaked nose” with narrow nasal tip, small mouth, horizontal crease of chin, bilateral earlobe hypoplasia, and clinodactyly of II toe and V finger bilaterally (right panel). (C) Images of individual 12 at the age of 5 years show a high anterior hairline, small upslanting palpebral fissures, thin eyebrows, a broad forehead, a short nose with anteverted nostrils, a long philtrum, and small mouth with thin lips and downturned corners. Note a somewhat short neck with low posterior hairline. (D) Images of individual 14 at the age of 19 years show upwardly slanted palpebral fissures, epicanthal folds, broad nasal bridge, long nose, downturned nasal tip, short columella, long philtrum, and thin upper vermillion. (E) Images of individual 15 at the age of 12 years show a high anterior hairline, a long nose with protruding columella hypoplastic alae nasi, thin upper lip vermilion, and low-set ears. (F) Images of individual 18 at the age of 3 years show midface flattening, flattened nasal bridge with rounded nasal tip, micrognathia, and a small mouth. (G) Images of individual 23 at the age of 20 months show a high anterior hairline, white and thin hair, absent eyelashes, small mouth, and micrognathia.

Figure 4

Analysis of DNA-binding affinity of selected BCL11B missense variants Luciferase assay utilizing HEK293T cells. (A) Firefly luciferase plasmid (containing a dELS within either SHANK3, LTBP3, or LTBP4 locus) and pNL1.1.PGK [Nluc/PGK] plasmid were transfected together with empty pRFP-N1, pRFP-N1-BCL11B-WT, or one of the depicted missense variants in pRFP-N1-BCL11B plasmid. Results are presented as the means from three independent experiments. Each experiment was normalized to the mean luminescence of measured samples to correct for different order of magnitude between experiments. (B) Experiments as in (A) performed for selected frameshift variants. (C) For analyses of potential dominant-negative effect, pEGFP-BCL11B-WT was added in combination with the above. Statistical significance was calculated by one-way ANOVA with recommended Dunnett correction for multiple comparisons (GraphPad Prism). Means ± standard deviation values are based on three independent experiments. Significantly different from BCL11B-WT: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; n.s., not significantly different from BCL11B-WT. (D) Experiments as in (C) performed for selected frameshift variants.

Figure 5

Modeling of BCL11B DNA-binding specificity due to BCL11B missense variants (A) Predicted DNA-binding sites of the BCL11B-WT for the four C2H2-ZnFs in which missense variants were identified. (B) Predicted DNA-binding sites for each of the missense variants within the α helix. Note that all missense variants affecting a “specificity residue” (shown in blue) alter the DNA-binding site and are therefore predicted to bind to different alternative genomic sequences as compared to the BCL11B-WT. Missense variants within the α helix not affecting a “specificity residue” (shown in violet) do not alter the DNA-binding specificity. Data are based on “Interactive PWM Predictor.”