Mutations in EBF3 Disturb Transcriptional Profiles and Cause Intellectual Disability, Ataxia, and Facial Dysmorphism

Abstract

From a GeneMatcher-enabled international collaboration, we identified ten individuals affected by intellectual disability, speech delay, ataxia, and facial dysmorphism and carrying a deleterious EBF3 variant detected by whole-exome sequencing. One 9-bp duplication and one splice-site, five missense, and two nonsense variants in EBF3 were found; the mutations occurred de novo in eight individuals, and the missense variant c.625C>T (p.Arg209Trp) was inherited by two affected siblings from their healthy mother, who is mosaic. EBF3 belongs to the early B cell factor family (also known as Olf, COE, or O/E) and is a transcription factor involved in neuronal differentiation and maturation. Structural assessment predicted that the five amino acid substitutions have damaging effects on DNA binding of EBF3. Transient expression of EBF3 mutant proteins in HEK293T cells revealed mislocalization of all but one mutant in the cytoplasm, as well as nuclear localization. By transactivation assays, all EBF3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene CDKN1A, and in situ subcellular fractionation experiments demonstrated that EBF3 mutant proteins were less tightly associated with chromatin. Finally, in RNA-seq and ChIP-seq experiments, EBF3 acted as a transcriptional regulator, and mutant EBF3 had reduced genome-wide DNA binding and gene-regulatory activity. Our findings demonstrate that variants disrupting EBF3-mediated transcriptional regulation cause intellectual disability and developmental delay and are present in ∼0.1% of individuals with unexplained neurodevelopmental disorders.

Keywords: EBF3; de novo mutation; developmental delay; gene regulation; intellectual disability; transcription factor.

Figure 1

EBF3 Mutations Identified in Ten Individuals with ID (A) Schematic representation of the exon-intron structure of EBF3. Black bars represent exons, and black lines represent introns. Mutations identified in the ID-affected individuals are indicated above the exon-intron structure. (B) Domain structure of EBF3, including the positions of the identified amino acid alterations. Amino acid numbers are given. Abbreviations are as follows: DBD, DNA-binding domain with an atypical zinc finger (ZNF; COE motif); IPT, Ig-like/plexins/transcription factors; HLH, helix-loop-helix motif; and TAD, transactivation domain. (C) Photographs of seven individuals show subtle yet distinct facial dysmorphism. All show a long face, tall forehead, high nasal bridge, deep philtrum, straight eyebrows, strabismus, short and broad chin, and mildly dysmorphic ears. Consent for the publication of photographs was obtained for the seven subjects.

Figure 2

Structural Impact of EBF3 Missense Mutations (A) Model of the DBD of an EBF3 monomer (cyan ribbon; affected residues are shown as sticks) bound to DNA (sticks). Hydrogen bonds are represented by yellow lines. Major interactions of affected residues are shown in (B)–(F). (B) Asn66 forms a hydrogen bond with a DNA phosphate group (left), which is disrupted by the substitution (right). In addition, the negative charge of aspartate at position 66 most likely leads to electrostatic repulsion of the phosphate group. (C) Gly171 is part of the protein-DNA interface (left). Substitution of Gly171 with the negatively charged asparagine could lead to an electrostatic repulsion of the DNA backbone (right). (D) Pro177 is closely localized to Asn174, which forms a hydrogen bond with the DNA (left), and to the zinc finger (right). Replacement of Pro177 with leucine could lead to a conformational change altering the position of Asn174 and possibly of the zinc finger, reducing the DNA-binding capacity of EBF3. His157 and Cys161, Cys164, and Cys170 are invariant residues coordinating Zn²⁺ (right). In-frame duplication of the amino acids His157, Glu158, and Ile159 could cause a conformational change of the zinc finger, reducing the DNA-binding capability of EBF3. (E) Arg209 forms hydrogen bonds with the backbone of Cys198 and Asn197, the latter of which forms a hydrogen bond with the DNA (left). Substitution at Arg209 leads to disruption of these hydrogen bonds, probably affecting the positioning of Asn197 (right). (F) Tyr141 is localized within a loop that is not directly involved in DNA binding but rather in EBF3 dimer formation (one EBF3 monomer is indicated by a pink ribbon, and the other is marked by a blue ribbon). Alteration of Tyr141 could lead to a conformational change at the dimer interface, probably resulting in reduced stability of the EBF3 dimer and interfering with its ability to bind to DNA.

Figure 3

EBF3 Mutants Show Impaired DNA Binding and Altered Subcellular Localization (A) HEK293T cells were transiently transfected with EBF3 expression constructs, fixed, treated with permeabilization-blocking solution, and incubated in mouse monoclonal anti-FLAG M2 antibody solution (1:200 dilution; clone F-3165, Sigma-Aldrich). After washing, cells were incubated with Alexa Fluor 488 coupled to goat anti-mouse IgG (1:1,000 dilution; ThermoFisher) and embedded in mounting solution (ProLong Diamond Antifade Mountant with DAPI, ThermoFisher). Cells were analyzed with the Olympus IX-81 epifluorescence microscope. Wild-type EBF3 (EBF3^WT, green) was exclusively localized in the nucleus (blue), whereas the DNA-binding-deficient mutant p.His157Ala (EBF3^H157A) and the disease-associated mutants p.Asn66Asp (EBF3^N66D), p.Tyr141Cys (EBF3^Y141C), p.Gly171Asp (EBF3^G171D), p.His157_Ile159dup (EBF3^H157_I159dup), p.Pro177Leu (EBF3^P177L), p.Arg209Trp (EBF3^R209W), and p.Arg303^∗ (EBF3^R303∗) were also located in the cytoplasm. Representative images are shown. Error bars represent 10 μm. (B) EBF3 mutants show impaired activation of luciferase reporter expression under the control of the CDKN1A (p21) promoter. HEK293T cells were transiently transfected with the expression construct(s) of interest, together with pGL2-p21 (CDKN1A) promoter-Luc and pREN in a 1:1:3 ratio of pREN(2 μg):pGL2-p21 promoter-Luc(2 μg):pFLAG-CMV4-EBF3 or pFLAG-CMV4-p53^WT(6 μg). pGL2-p21 promoter-Luc encodes Photinus luciferase, and pREN is a derivate of the pFiRe-basic encoding Renilla luciferase. Wild-type p53 was used as an internal control. Dual luciferase assays were done with the extracts of transfected cells 48 hr after transfection. Data were normalized to the activity of Renilla luciferase, and basal promotor activity for transfection with pFLAG-CMV4-cassetteA (control vector) was considered to be 1. Compared to the empty vector (control; white bar), expression of EBF3^WT (green bar) and p53 (black bar) led to 4- to 5-fold elevated promoter activity. The DNA-binding-deficient EBF3^H157A mutant (yellow bar) and all disease-associated EBF3 mutants (blue bars) showed strongly reduced or no activation of the luciferase reporter. The normalized luciferase activity (mean ± SD) of three independent experiments is depicted as the fold induction relative to that of cells transfected with a control vector. All comparisons are in reference to EBF3^WT, and p values were calculated with the two-sided Student’s t test (^∗∗p < 0.005, ^∗∗∗p < 0.0005). (C) EBF3 mutants are not tightly bound to chromatin. 24 hr after transfection of HEK293T cells with EBF3 expression constructs or pFLAG-CMV4-cassetteA (control vector), in situ subcellular fractionation was performed. Cells were incubated with CSK buffer containing 0.1% Triton-X. The cytoplasmic extracts were removed, and proteins were precipitated. Cells were subsequently treated with CSK buffer supplemented with 0.5% Triton-X. Nuclear extracts were removed, and proteins were precipitated. Total cell lysate (TCL), cytoplasmic fraction (CF), and nuclear fraction (NF) were analyzed by SDS-PAGE and immunoblotting with a mouse monoclonal anti-FLAG M2 peroxidase conjugate (1:50,000 dilution; Sigma-Aldrich). For control of equal loading, TCL was analyzed with mouse anti-GAPDH antibody (1:10,000 dilution; Abcam). The mutant EBF3 proteins were present in both the CF and NF. In marked contrast, EBF3^WT was present in only minimal amounts in the NF, demonstrating exclusive nuclear localization and strong chromatin binding. Data represent four independent experiments.

Figure 4

EBF3^P177L Overexpression Shows Less Transcriptome Alteration and Whole-Genome EBF3 Occupancy Than EBF3^WT (A) Expression of EBF3^P177L has less transcriptome alteration than EBF3^WT. We generated two SK-N-SH (ATCC HTB-11) EBF3^WT- and two EBF3^P177L-expressing stable cell-line seed stocks, each of which represented a pool of two transfections (1 × 10⁶ SK-N-SH cells transfected with 5 μg of respective overexpression construct). Replicates from these samples were grown and maintained independently under selection (along with SK-N-SH controls), and then RNA was isolated and used to make sequencing libraries. RNA-seq libraries were prepared with the Nextera DNA Library Sample Prep Kit according to established protocols. Libraries were sequenced on an Illumina HiSeq 2500. All statistical analyses were performed in R (v.3.2.1). RNA-seq reads were processed with aRNApipe. To perform differential gene-expression analysis, we used the R DESeq2 package (v.1.8.2) with default Wald-test hypothesis testing with an adjusted p value (FDR) cutoff of 0.05. GO term enrichment was performed with the online tool g:Profiler with all GO term annotation categories. The Venn diagram depicts genes identified as significantly differentially expressed between SK-N-SH cells (control) and EBF3^WT and EBF3^P177L cells; 154 were shared between EBF3^WT and EBF3^P177L cells. (B) A heatmap of DESeq2 variance-stabilized RNA-seq expression values compares SK-N-SH control (CTL), EBF3^WT, and EBF3^P177L samples for genes determined to be significantly different between either EBF3^WT or EBF3^P177L cells and control SK-N-SH cells. (C) EBF3^P177L reduces genome-wide EBF3 binding sites determined by ChIP-seq (bottom). Available anti-EBF3 antibodies have been found to have some degree of cross-reactivity with other EBF family members, which limits the interpretability of specific family member binding sites. Therefore, we performed ChIP by using a high-affinity monoclonal anti-FLAG M2 antibody (Sigma) that targets the C-terminal 3X-FLAG epitope of the EBF3 cDNA constructs. Libraries were sequenced on an Illumina NextSeq. ChIP-seq reads were aligned with the Burrows-Wheeler Aligner to the UCSC Genome Browser (hg19), and peaks were identified for each replicate with MACS2.1.0 with an -mfold cutoff of [10, 30]. We merged replicate overlapping peaks with BEDTools to generate the final peak lists used in downstream analyses. The most significant motifs with centrally enriched distribution for EBF3^WT and EBF3^P177L were identified with MEME-Suite (top). (D) Significantly upregulated genes are closer to EBF3^WT and EBF3^P177L binding sites. A cumulative distribution function (CDF) plot of EBF3^WT and EBF3^P177L shows the distance from the GRCh37 TSS to the nearest EBF3^WT and EBF3^P177L binding sites for genes identified as upregulated or downregulated (FDR < 0.05, log₂ fold change > 0 and < 0, respectively) in comparison to background. (E) EBF3^WT or EBF3^P177L significantly differentially expressed genes with a TSS within 50 kb of shared ChIP-seq binding sites exhibit a relatively greater absolute log₂ fold change in EBF3^WT than the same genes in EBF3^P177L expression data. Linear regression of log₂ fold changes for these genes (n = 146) exhibits a downward-skewed slope of 0.72 (blue line) in comparison to the null expectation of perfect correspondence (slope = 1, dashed red line), indicating comparatively reduced alteration of expression for significant EBF3^WT genes by EBF3^P177L.