Supplementary Information 1 SUPPLEMENTARY INFORMATION Supplementary Information 2 Supplementary Figure 1 Supplementary Figure 1 (a) Sequence electropherograms show the EBF3 c.625C>T mutation in DNA isolated from leukocytes and fibroblasts of the two affected siblings (subjects 1 and 2) in the heterozygous state (top rows). Sanger sequencing demonstrated mosaicism of the c.625C>T mutation in leukocyte-derived DNA of the mother. The mutation was not visible in the sequence derived from her buccal cell-derived DNA (third row). The healthy family members showed wild-type sequence in leukocyte- derived DNA. Sanger traces show the c.1101+1G>T, c.530C>T and c.469_477dup mutations in leukocyte-derived DNA of subjects 5, 6, and 10 (left bottom row), respectively, and wild-type sequence in their parents (right bottom rows). Arrows point to the position of the mutations. (b) Cloning of exon 7-containing amplicons, followed by colony PCR and sequencing revealed that ~18% of leukocytes and ~4% of buccal cells of the mother contain the heterozygous EBF3 mutation (9% and 2% of clones with the mutated allele). (c) Sequence electropherograms show heterozygosity of the c.625C>T mutation in fibroblast-derived cDNA of subjects 1 and 2. Supplementary Information 3 Supplementary Figure 2 66 EBF3_human 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_chimpanzee 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_macaque 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_mouse 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_rat 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSN EBF3_zebrafish 50 ARAHYEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKEKEPNSEKTNNGIHYKLQLLYSN EBF3_fruit fly 66 GRAHFEKQPPSNLRKSNFFHFVIALYDRAGQPIEIERTAFIGFIEKDSESDATKTNNGIQYRLQLLYAN EBF3_mosquito 6 GRAHFEKQPPSNLRKSNFFHFVVALYDRAGQPIEIERTAFIGFIEKDQEPDGQKTNNGIQYRLQLLYAN EBF3_clawed frog 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVEKDKEPNSEKTNNGIHYKLQLLYSN EBF1_human 50 ARAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVGFVEKEKEANSEKTNNGIHYRLQLLYSN EBF2_human 49 SRAHFEKQPPSNLRKSNFFHFVLALYDRQGQPVEIERTAFVDFVENDKEQGNEKTNNGTHYKLQLLYSN 141 157 171 177 EBF3_human 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_chimpanzee 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_macaque 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_mouse 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_rat 119 GVRTEQDLYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_zebrafish 119 GVRTEQDLYVRLIDSMTKQAIIYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_fruit fly 135 GARQEQDIFVRLIDSVTKQAIIYEGQDKNPEMCRVLLTHEVMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_mosquito 75 GARQEQDIFVRLIDSVTKQAIVYEGQDKNPEMCRVLLTHEVMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF3_clawed frog 119 GVRTEQDLYVRLIDSMTKQAIIYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF1_human 119 GIRTEQDFYVRLIDSMTKQAIVYEGQDKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFF EBF2_human 118 GVRTEQDLYVRLIDSVTKQPIAYEGQNKNPEMCRVLLTHEVMCSRCCEKKSCGNRNETPSDPVIIDRFF COE/ZNF 209 243 EBF3_human 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_chimpanzee 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_macaque 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_mouse 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_rat 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_zebrafish 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF3_fruit fly 204 LKFFLKCNQNCLKNAGNPRDMRRFQVVISTQVAVDGPLLAISDNMFVHNNSKHGRRAKRLDTTE EBF3_mosquito 144 LKFFLKCNQNCLKNAGNPRDMRRFQVVIATQVAVDGPLLAISDNMFVHNNSKHGRRAKRLD-PE EBF3_clawed frog 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF1_human 188 LKFFLKCNQNCLKNAGNPRDMRRFQVVVSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE EBF2_human 187 LKFFLKCNQNCLKTAGNPRDMRRFQVVLSTTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSE Supplementary Figure 2 Amino acid sequence alignment of the human EBF3 DNA-binding domain (aa 50-251; NP_001005463) with orthologs and two paralogs [human EBF1 (NP_001277289) and human EBF2 (NP_073150)], showing conservation of amino acids N66, Y141, H157, E158, I159, G171, P177, R209 and R243 between species and within the human EBF family (EBF1, EBF2 and EBF3). Multiple alignments were gathered from http://www.ncbi.nlm.nih.gov/homologene/; alignment with EBF1 and EBF2 was performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Conserved residues mutated in the patients are highlighted in red; arginine 243 altered in tumors is highlighted in grey. The amino acid stretch constituting the atypical zinc finger (COE/ZNF) is indicated by a black line (aa 157-170). Supplementary Information 4 Supplementary Figure 3 Supplementary Figure 3 (a) Model of the DNA-binding domain of an EBF3 monomer (cyan ribbon, affected residues 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 panel), which is disrupted by the mutation (right panel). In addition, the negative charge of aspartate at position 66 likely leads to electrostatic repulsion of the phosphate group. (c) Gly171 is part of the protein-DNA interface (left panel). Mutation of Gly171 to the negatively charged asparagine could lead to an electrostatic repulsion of the DNA backbone (right panel). (d) Pro177 is localized in close proximity to Asn174, which forms a hydrogen bond with the DNA (left panel), and to the zinc finger (right panel). Replacement of Pro177 by leucine may lead to a conformational change altering the position of Asn174 and possibly of the zinc finger, reducing the DNA-binding capacity of EBF3. His157 together with Cys161, Cys164 and Cys170 are invariant residues coordinating Zn2+ (right panel). In-frame duplication of the amino acids His157, Glu158 and Ile159 may cause a conformational change of the zinc finger reducing DNA-binding capability of EBF3. (e) Arg209 forms hydrogen bonds with the backbone of Cys198 and Asn197, with the latter forming a hydrogen bond with the DNA (left panel). Mutation of Arg209 leads to disruption of these hydrogen bonds, probably affecting the positioning of Asn197 (right panel). (f) Tyr141 is localized within a loop which is not directly involved in DNA-binding but in EBF3 dimer formation (pink ribbon). Mutation of Tyr141 may lead to a conformational change at the dimer interface, resulting in reduced stability of the EBF3 dimer and interfering with its ability to bind to DNA. Supplementary Information 5 Supplementary Figure 4 Supplementary Figure 4 Co-expression of wild-type EBF3 and EBF3 mutants in transactivation assays. (a) HEK 293T cells were transfected with EBF3 mutant-expressing vectors together with a plasmid expressing wild-type EBF3 (1:1) or with empty vector alone (control). Expression of only wild-type EBF3 (green bar) led to a 4- to 5-fold elevated promoter activity compared with cells transiently transfected with empty vector (white bar). Co-expression of EBFWT H157A and the DNA binding-deficient EBF3 mutant (yellow bar) or either of the disease-associated EBF3 mutants N66D, Y141C, Q305*, H157_I159dup and R303* (blue bars) resulted in ~50% reduction of luciferase reporter activity. The three mutants P177L, R209W and G171D did not cause any significant reduction in reporter activity. The normalized luciferase activity (mean ± s.d.) of three independent experiments is depicted as the fold induction relative to cells transfected with a control vector. All comparisons are in reference to wild-type EBF3, and P values were calculated N66D Q305* using the two-sided Student’s t test. (b) Increasing amounts of EBF3 (left panel; blue bars) and EBF3 (right panel; blue bars) reduce promoter activity indicating a dominant-negative effect on wild-type EBF3. HEK 293T cells were transiently transfected with a constant amount of wild-type EBF3 (4 µg; +), together with the reported amount of mutant EBF3, with 8 µg wild-type EBF3 (++; green bar) or with empty vector alone (-; white bar). Dual luciferase assays were done with the extracts of transfected cells 48 hours after transfection. The normalized luciferase activity (mean ± s.d.) of three independent experiments is depicted as the fold induction relative to cells transfected with a control vector. For all comparisons, P values were calculated using the two-sided Student’s t test. *P <0.05, **P < 0.005, ***P < 0.0005; n.s.: not significant. Supplementary Information 6 Supplementary Figure 5 Supplemental Figure 5 Boxplots of variance stabilized counts for significantly differentially expressed genes (FDR <0.05) between untransfected SK-N-SH cells (blue), EBF3WT (green), and EBF3P177L (red) expressing cells. Supplementary Information 7 Supplementary Figure 6 Supplementary Figure 6 Gene Ontology (GO) term analysis of genes significantly differentially expressed between EBF3WT and EBF3P177L versus SK-N-SH control. (a) EBF3WT top 10 most significantly enriched GO terms by p-value for all GO term categories. (b) EBF3P177L top 10 most significantly enriched GO terms by p-value for all GO term categories (BP: Biological Processes, CC: Cellular Compartment). Supplementary Information 8 Supplementary Figure 7 Supplementary Figure 7 Schematic of EBF3WT and EBF3P177L