Mutations in ZBTB20 cause Primrose syndrome

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Jul 13, 2014 - muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the ...
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Mutations in ZBTB20 cause Primrose syndrome Viviana Cordeddu1,18, Bert Redeker2,18, Emilia Stellacci1, Aldo Jongejan3, Alessandra Fragale4, Ted E J Bradley5, Massimiliano Anselmi6, Andrea Ciolfi1, Serena Cecchetti7, Valentina Muto1, Laura Bernardini8, Meron Azage9, Daniel R Carvalho10, Alberto J Espay11, Alison Male12, Anna-Maja Molin13, Renata Posmyk14, Carla Battisti15, Alberto Casertano16, Daniela Melis16, Antoine van Kampen3, Frank Baas5, Marcel M Mannnens17, Gianfranco Bocchinfuso6, Lorenzo Stella6, Marco Tartaglia1,19 & Raoul C Hennekam2,17,19 Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism. Many mendelian disorders with isolated or syndromic short stature have been recognized, but the spectrum of conditions with increased growth appears much more restricted1. Among the latter, the 3q13.31 microdeletion (del3q13.31) syndrome (MIM 615433) is a multisystem disorder characterized by increased postnatal growth, hypotonia, intellectual disability, disturbed behavior and unusual facial features2,3. The recurrent deletion in this syndrome encompasses 28 genes, with a minimum region containing 5 RefSeq genes (DRD3, ZNF80, TIGIT, MIR568 and ZBTB20)2,3. Primrose syndrome (MIM 259050) is a condition characterized by increased growth of the brain and greater body height compared to the general population, hypotonia, intellectual disability, autism and other behavioral concerns4,5. Facial signs resemble those in the del3q13.31

syndrome (Fig. 1a). Individuals with Primrose syndrome also develop diabetes in adulthood, progressive muscle wasting, hearing loss and ectopic calcifications. We analyzed four unrelated subjects with Primrose syndrome and the unaffected parents of two of these subjects by whole-exome sequencing (Online Methods). On the basis of the sporadic occurrence of this syndrome and the absence of consanguinity, a de novo variant was assumed to be the most likely cause. Analysis of whole-exome sequencing data documented that each proband was heterozygous for three to six previously unannotated, functionally relevant variants shared by one or more other probands, but only the variants in ZBTB20 were present in all probands. Similarly, in the 2 family trios, 4 and 15 genes were found to have a putatively de novo variant in the 2 sporadic cases, but only ZBTB20 had de novo variants shared by the 2 affected individuals (Supplementary Table 1). Sanger sequencing validated all the ZBTB20 variants, and parental genotyping demonstrated the de novo origin of the variants (Supplementary Fig. 1). For two subjects, DNA from various tissues was available, and all tissues analyzed harbored the ZBTB20 mutation, indicating the presence of the mutation early in embryonic development, with it likely being of germline origin. Mutation analysis was subsequently performed on four additional affected subjects, identifying a ZBTB20 mutation in each and supporting genetic homogeneity (Table 1). ZBTB20 resides in the del3q13.31 syndrome critical region and encodes a transcriptional repressor of the broad complex tramtrack bric-a-brac (BTB) zinc-finger family6,7. ZBTB20 has a role in neurogenesis, glucose metabolism and postnatal growth 8–10. It is characterized by an N-terminal BTB domain involved in protein-protein interaction and five C2H2 zinc fingers at the C terminus, mediating protein binding to regulatory sites within promoters (Fig. 1b). All affected codons in Primrose syndrome were conserved among orthologs (Supplementary Fig. 2), and involved residues were located in the first (Lys590, Gln591 and His596) and second (Leu621 and Val626) zinc fingers and in the linker connecting these motifs (Thr601, Gly602 and Lys604). Among these residues, His596 coordinates the Zn2+ ion; it is invariably conserved among C2H2 zinc fingers (SMART alignment, ZnF_C2H2), and its replacement was predicted to disrupt domain structure. The structural impact and functional effect of the mutations were explored using a homology model for the

1Dipartimento

di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy. 2Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. 3Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. 4Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Rome, Italy. 5Department of Genome Analysis, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. 6Dipartimento di Scienze e Tecnologie Chimiche, Università ‘Tor Vergata’, Rome, Italy. 7Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy. 8Laboratorio Mendel, Fondazione Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy. 9Department of Medical Genetics, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA. 10Medical Genetic Unit, SARAH Network of Rehabilitation Hospitals, Brasilia, Brazil. 11Department of Neurology, University of Cincinnati, Gardner Family Center for Parkinson’s Disease and Movement Disorders, Cincinnati, Ohio, USA. 12Clinical Genetics Department, Great Ormond Street Hospital for Children National Health Service (NHS) Foundation Trust, London, UK. 13Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden. 14Podlaskie Center of Clinical Genetics, Białystok, Poland. 15Dipartimento di Scienze Neurologiche, Neurochirurgiche e del Comportamento, Università degli Studi di Siena, Policlinico Le Scotte, Siena, Italy. 16Dipartimento di Pediatria, Facoltà di Medicina e Chirurgia, Università ‘Federico II’, Naples, Italy. 17Department of Pediatrics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. 18These authors contributed equally to this work. 19These authors jointly directed this work. Correspondence should be addressed to M.T. ([email protected]) or R.C.H. ([email protected]). Received 31 January; accepted 23 June; published online 13 July 2014; doi:10.1038/ng.3035

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B r i e f c o m m u n i c at i o n s a

c Leu621

His596 Val626 Lys604

Lys604 Gln591

Gly602 His596

b

Leu621 Thr601

G602A T601I K604T H596R L621F Q591E V626M K590Q

Gly602 Gln591

Lys590 Lys590

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Thr601

Figure 1  ZBTB20 is mutated in Primrose syndrome. (a) Clinical features of the affected subjects. Note wide forehead, ptosis, downslanting palpebral fissures, large jaw, calcified pinnae (arrows), camptodactyly and truncal obesity. Permission was obtained to publish pictures of all subjects (12D0186, 11D5407, 11D5135, 11D5028 and KND_01). (b) ZBTB20 domain structure (cyan box, BTB domain; yellow boxes, zinc fingers) and location of affected residues. (c) Model of DNA-bound ZBTB20 zinc fingers 1–4 (ribbon; helices in red, β strands in yellow). Relevant residues (sticks), DNA backbone (gold ribbon), bases (sticks) and phosphates (dotted spheres; in addition, those interacting with ZBTB20 are shown as sticks) are shown. Hydrogen bonds and electrostatic interactions are represented by green and blue dashed lines, respectively. Major interactions of affected residues are shown in lateral panels. Lys590 and Lys604 form a salt bridge with a DNA phosphate group, disrupted by the mutations. The amino group of Gln591 forms hydrogen bonds with a phosphate oxygen molecule and a DNA base; p.Gln591Glu introduces repulsion with the DNA backbone. His596 is an invariant residue coordinating Zn2+ and forms a hydrogen bond with a DNA phosphate group. Thr601 and Gly602 belong to the conserved TGEKP motif. Thr601 forms a hydrogen bond with the amino group of Glu603; Gly602 favors the peculiar left-handed backbone conformation. Leu621 is part of the hydrophobic cluster surrounding His606, whereas Val626 forms hydrophobic contacts with residues of the third zinc finger (Thr644, hydrogen bonded with DNA) and linker (Val631).

DNA-bound conformation of zinc fingers 1–4 (residues 576–684) with the nucleotide stretch of the promoter for AFP (encoding α-fetoprotein) known to contain a ZBTB20 recognition site11. Four of the eight alterations were predicted to directly affect DNA binding (Fig. 1c). Similarly, substitutions of Thr601 and Gly602, which belong to the conserved TGEKP motif controlling the correct orientation of the linker connecting two adjacent zinc fingers12, were expected to destabilize the protein-DNA complex13,14. Leu621 was not predicted to interact directly with DNA or with the linker region. Substitution of homologous residues in zinc fingers, however, can markedly affect binding affinity for DNA15, and molecular dynamics simulations performed on the second zinc finger (residues 606–633) indeed predicted an indirect effect on DNA binding affinity due to a substantial rearrangement of His606 (Supplementary Fig. 3), which contributes to stabilizing the linker region12. Similarly, the p.Val626Met substitution

was expected to perturb a hydrophobic cluster that stabilizes the mutual arrangement of the second and third zinc fingers. In transient transfection experiments in HEK 293T cells, all mutants were efficiently expressed (data not shown). Confocal microscopy ana­ lysis confirmed the nuclear localization of wild-type ZBTB20 and all tested mutants, even though a distinctive, non-homogeneous distribution pattern, suggestive of protein aggregation, was observed with the mutants (Supplementary Fig. 4). Treatment with CSK buffer, which was used to selectively remove free protein aggregates from the nucleus, indicated that the mutants loosely interacted with chromatin, in contrast to what was observed for the wild-type protein (Supplementary Fig. 4 and Supplementary Table 2). DNA binding assays, using biotinylated oligonucleotides encompassing the AFP promoter minimal responsive sequence, demonstrated strongly reduced DNA binding for all the mutants tested (Supplementary Fig. 5a,

Table 1  List of identified ZBTB20 mutations in individuals with Primrose syndrome Subject 11D5407 12D0186 KND_01 11D5135 12D6966 12_58589 11D5028 PRS_02

Nucleotide changea

Coding exon

Amino acid changea

Protein domain

Origin

Functional impactb (pph2_prob/SIFT score)

c.1768A>C c.1771C>G c.1787A>G c.1802C>T c.1805G>C c.1811A>C c.1861C>T c.1876G>A

3 3 3 3 4 4 4 4

p.Lys590Gln p.Gln591Glu p.His596Arg p.Thr601Ile p.Gly602Ala p.Lys604Thr p.Leu621Phe p.Val626Met

ZnF I ZnF I ZnF I Linker, ZnF I–II Linker, ZnF I–II Linker, ZnF I–II ZnF II ZnF II

De novo De novo, Germline De novo, Germline De novo De novo De novo De novo Not testedc

0.998/0.01 0.924/0.02 0.991/0.00 0.999/0.00 0.999/0.01 0.999/0.00 0.999/0.03 0.998/0.11

ZnF, zinc finger. aNucleotide

and amino acid positions refer to the longer cDNA and protein isoforms (NM_001164342.1; NP_001157814.1). bPrediction based on PolyPhen-2 and SIFT. cParental DNA was not available for molecular analyses.



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B r i e f c o m m u n i c at i o n s left), which correlated with their strongly reduced ability to repress transcription of a reporter gene under the control of the same promoter sequence (Supplementary Fig. 5b,c). Comparable results were obtained using the untruncated AFP promoter in transiently transfected COS1 and HepG2 cells (data not shown). Reduced levels of DNA-bound ZBTB20 and less efficient AFP promoter repression were also observed in cells coexpressing the wild-type protein in the presence of each of the disease-causing mutants (Supplementary Fig. 5a–c), consistent with the mutations having a dominant-negative impact on the wild-type allele. Individuals positive for ZBTB20 mutation exhibited a consistent phenotype characterized by increased growth, variable intellectual disability, autistic traits and other behavioral problems, hypotonia and distinctive facial features overlapping with the phenotype of the del3q13.31 syndrome (Supplementary Fig. 6 and Supplementary Table 3). The phenotype in Primrose syndrome, however, is more severe, which is in line with the present data suggesting a dominantnegative impact of the underlying mutations on the wild-type protein in Primrose syndrome compared to the haploinsufficiency for ZBTB20 functions that likely underlies the del3q13.31 syndrome. Specifically, glucose metabolism was invariably disturbed in subjects with Primrose syndrome, with insulin-resistant diabetes occurring in adulthood, a finding consistent with the function of ZBTB20 in controlling β cell function and glucose homeostasis, which are hypothesized to be mediated by various mechanisms8,9,11. Distal muscle wasting leading to progressive contractures in adulthood was also a recurrent finding. Affected individuals exhibited hearing loss and ectopic calcification of the ears and brain during puberty or early adulthood. Consistent with the impaired functioning of the mutants and the repressive role of ZBTB20 on AFP transcription11, all tested individuals exhibited increased AFP expression. One subject with Primrose syndrome developed cancers5. Whereas Zbtb20 knock-in mouse models are not available for comparison with the present data, Zbtb20−/− mice were reported to have abnormal hippocampal development, behavioral problems, growth retardation and reduced body mass, high and constitutive AFP expression throughout adult life, metabolic dysfunction and premature death8,10,11. None of these features were observed in heterozygous mice, indicating the absence of an obvious gene dosage effect. Of note, in contrast to what is observed in Primrose syndrome, Zbtb20−/− mice display hypoglycemia, increased insulin sensitivity and energetic deficit8. By contrast, β cell–specific targeted disruption of Zbtb20 has been demonstrated to cause hyperglycemia, hypoinsulinemia, glucose intolerance and impaired glucose-stimulated insulin secretion9, strongly indicating a complex role for ZBTB20 in controlling glucose metabolism at multiple levels. Overall, we report that heterozygous ZBTB20 mutations affecting the DNA-binding domain of the transcription factor underlie Primrose syndrome, possibly through dominant-negative action. Our findings establish a genetic link between this disorder and the clinically related del3q13.31 syndrome, and they delineate the impact of ZBTB20 functional dysregulation and haploinsufficiency on development, growth and metabolism.

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URLs. Online Mendelian Inheritance in Man (OMIM), http://www. ncbi.nlm.nih.gov/omim/; NCBI Gene, http://www.ncbi.nlm.nih. gov/gene/; Picard tools, http://picard.sourceforge.net/; wAnnovar, http://wannovar.usc.edu/; dbSNP137, http://www.ncbi.nlm.nih. gov/projects/SNP/snp_summary.cgi/; 1000 Genomes Project, http:// www.1000genomes.org/; HapMap Project, http://hapmap.ncbi.nlm. nih.gov/; SMART, http://smart.embl-heidelberg.de/; Protein Data Bank (PDB), http://www.rcsb.org/pdb/home/home.do. Methods Methods and any associated references are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We are grateful to the patients and their families. We thank M.L. Motta and S. Venanzi (Istituto Superiore di Sanità) for technical assistance, the CINECA Consortium for computational resources, and K. Riabowol (University of Calgary), H. Nakabayashi (Hokkaido Information University) and Y. Miura (Graduate School of Medical Science, Nagoya, Japan) for providing the AFP promoter luciferase construct and the HNF1 and ATBF1 expression vectors, respectively. This work was supported by funding from the Istituto Superiore di Sanità (RC2013) to M.T. AUTHOR CONTRIBUTIONS V.C. and B.R. performed exome sequencing data validation and mutation analysis and wrote the manuscript. A.J., T.E.J.B., A. Ciolfi, A.v.K., F.B. and M.M.M. carried out the exome sequencing data processing and analyses. E.S., A.F., V.M. and L.B. performed the functional studies and genotyping. M. Anselmi, G.B. and L.S. designed and performed the molecular dynamics simulations and structural analyses. S.C. performed the confocal laser scanning microscopy analysis. M. Azage, D.R.C., A.J.E., A.M., A.-M.M., R.P., C.B., A. Casertano, D.M. and R.C.H. recruited and clinically characterized the study subjects and collected blood samples. M.T. and R.C.H. conceived the project, designed and supervised the experiments, analyzed and interpreted the data, and wrote the manuscript. All authors contributed to the final manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Sabin, M.A. et al. Best Pract. Res. Clin. Endocrinol. Metab. 25, 207–220 (2011). 2. Molin, A.M. et al. J. Med. Genet. 49, 104–109 (2012). 3. Shuvarikov, A. et al. Hum. Mutat. 34, 1415–1423 (2013). 4. Primrose, D.A. J. Ment. Defic. Res. 26, 101–106 (1982). 5. Mathijssen, I.B. et al. Eur. J. Med. Genet. 49, 127–133 (2006). 6. Zhang, W. et al. Biochem. Biophys. Res. Commun. 282, 1067–1073 (2001). 7. Mitchelmore, C. et al. J. Biol. Chem. 277, 7598–7609 (2002). 8. Sutherland, A.P.R. et al. Mol. Cell. Biol. 29, 2804–2815 (2009). 9. Zhang, Y. et al. Gastroenterology 142, 1571–1580 (2012). 10. Xie, Z. et al. Proc. Natl. Acad. Sci. USA 107, 6510–6515 (2010). 11. Xie, Z. et al. Proc. Natl. Acad. Sci. USA 105, 10859–10864 (2008). 12. Wolfe, S.A. et al. Annu. Rev. Biophys. Biomol. Struct. 29, 183–212 (2000). 13. Choo, Y. & Klug, A. Nucleic Acids Res. 21, 3341–3346 (1993). 14. Wuttke, D.S. et al. J. Mol. Biol. 273, 183–206 (1997). 15. Dhanasekaran, M. et al. Biochemistry 47, 11717–11724 (2008).



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Subjects. Eight subjects with a phenotype fitting Primrose syndrome were included in the study. To our knowledge, this cohort includes all available individuals diagnosed with this disorder. Among them, five had previously been described in detail5,16–19. Clinical features are summarized in Supplementary Table 3. DNA specimens were collected following institutional review board–approved protocols (Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Facoltà di Medicina e Chirurgia, Università ‘Federico II’, Naples, Italy; Dipartimento di Scienze Neurologiche, Neurochirurgiche e del Comportamento, Università degli Studi di Siena, Siena, Italy). Informed consent for DNA storage and genetic analyses was obtained from all subjects. Exome sequencing and sequence data analysis. Targeted enrichment and massively parallel sequencing were performed on genomic DNA extracted from circulating leukocytes. Enrichment of the whole exome was performed using NimbleGen SeqCap EZ Library v. 3.0 (Roche). Each captured library was then loaded onto the HiSeq 2000 platform (Illumina) (KND_01 and unaffected parents) or SOLiD550xl platform (Applied Biosystems) (11D5028 and unaffected parents, 11D5135 and 12D0186). For the former, raw image files were processed by CASAVA 1.7 (Illumina) for base calling with default parameters. Paired-end reads were aligned to the human genome (UCSC GRCh37/hg19) with the Burrows-Wheeler Aligner (BWA V. 0.7.5a)20. For the latter, pairedend and single-end sequence reads were aligned to hg19 using the Lifescope aligner (v2.5.1) (Applied Biosystems). Presumed PCR duplicates were discarded using Picard tools (see URLs) or Lifescope. Local realignment and base quality score recalibration were performed with the Genome Analysis Toolkit (GATK2)21. After mapping reads to the human genome reference, mean target region coverage was 97.4%, with average sequencing depth on target of 92×. SNPs and small indels were identified by means of the GATK HaplotypeCaller and UnifiedGenotyper algorithms22. Variants with a quality score of >50 and a quality-by-depth score of >1.5 were retained; variants with values below these thresholds or resulting from four or more reads having ambiguous mapping (this number being >10% of all aligned reads) were discarded. Variants were filtered against available public (1000 Genomes Project and dbSNP138) and in-house databases. An average of 4,068 previously unreported variants were identified that were filtered to retain 139 to 393 sequence changes located in exons, having any functional effect, and splice sites (intronic variants at exonintron junctions ranging from −5 to +5). Functional annotation of variants was performed using SnpEff v3.4 and dbNSFP23,24. Variant validation and mutation analysis. Sequence validation and segregation analyses for all the candidate variants, as well as mutation scanning of the entire ZBTB20 coding sequence (NM_001164342.1), were performed by Sanger sequencing. Primer pairs designed to amplify the ZBTB20 coding exons and their intron boundaries (NC_000003.12, 114,033,348–114,866,132, complement) are listed in Supplementary Table 4. Paternity was confirmed by STR genotyping (PowerPlex ESX16 System, Promega). Structural analyses. The three-dimensional structure of the first four zincfinger domains of ZBTB20 (residues 576–684) was initially obtained by means of homology modeling using the SWISS-MODEL web-based service 25. The crystallographic structure at 1.96-Å resolution of Aart, containing six zinc fingers in complex with DNA (PDB entry 2i13, chain A), was used as the template26. The selected template ensured the best homology score (44%) on the longest amino acid sequence. The four zinc fingers were initially modeled in distinct pairs, so that the last zinc finger in a given pair was the first in the following one. Chunks were then linked together, aligning the zinc-finger pairs over the backbone of their common zinc finger, resulting in no substantial differences between models. The coordinates of the DNA

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backbone were taken from the crystal structure of the double-stranded DNA bound to Aart26. The DNA bases were then replaced using COOT27 according to the nucleotide stretch of the human AFP promoter used in the DNAbinding and transactivation experiments. ZBTB20 was associated with the AGTAACAGATAT sequence, which had the highest score in the Zinc Finger Tools server28. The structure of the protein-DNA complex was refined using the GROMACS version 4.6 software package29. The DNA–zinc finger complex was modeled using the AMBER99SB Force Field30 augmented with the Zinc AMBER Force Field (ZAFF)31. Protein termini were considered to be capped. After an energy minimization in implicit solvent, performed while restraining DNA atomic positions with harmonic potentials, the system was solvated in a bath of TIP3P32 water molecules, with 36 Na+ ions added to compensate for the net negative charge of the protein-DNA complex. The solvent was relaxed by energy minimization while restraining the protein-DNA complex atomic positions. After a molecular dynamics simulation of 5 ns, in which the system temperature was brought to 300 K, 200 simulated annealing cycles of 50 ps each were performed, using 2 independent annealing schedules for protein and water (heated to 350 K and 450 K, respectively). During simulated annealing, the DNA coordinates were kept fixed by holonomic constraints, whereas no additional restraints were imposed on the protein. Molecular dynamics simulations and simulated annealing were performed in a constant volume box of 6.2 × 10.7 × 6.8 nm3, and the LINCS method was applied to constrain covalent bond lengths33, allowing an integration step of 2 fs. Electrostatic interactions were calculated with the Particle-Mesh Ewald method34. The temperature was controlled by separately coupling protein, DNA and solvent to an external temperature bath35. After simulated annealing, the structure with the lowest sum of protein-DNA binding energy and protein conformational energy was selected. Next, an energy minimization was performed in explicit solvent, using a conjugate gradient algorithm with flexible water molecules and keeping the DNA coordinates fixed by position restraints. Of note, the model was validated against the corresponding sequence of the mouse Afp promoter (AGTCATATGTTT), located within the nucleotide stretch demonstrated to contain a ZBTB20-binding site8. The mouse sequence retained the main contacts with the protein and, as a consequence, has a comparable protein-DNA binding energy. Molecular dynamics simulations. To analyze the structural effects of the diseasecausative p.Leu621Tyr amino acid substitution, molecular dynamics simulations were performed on the isolated second zinc finger, free in solution. Both the wild-type domain and the Leu621Tyr mutant were simulated. Initial coordinates were taken from the model, and the termini of the domains were capped. Each domain was solvated in a cubic box with TIP3P water molecules32. After a short equilibration, productive simulations of 500 ns were carried out, according to the protocol described above, at constant temperature (300 K) and pressure (1 bar). Functional studies. HEK 293T (human embryonic kidney) (CRL-11268, American Type Culture Collection), COS1 (monkey kidney) (1650, American Type Culture Collection) and HepG2 (human hepatoma) (provided by A. Ruggieri, Istituto Superiore di Sanità, Rome) cells were maintained in low-glucose DMEM with 10% FBS and supplements (Euroclone). Cells were authenticated by STR profiling and tested negative for mycoplasma contamination. The disease-causative nucleotide substitutions were introduced into human ZBTB20 cDNA cloned into the pCDNA6-Xpress vector (KpnIXbaI sites; encoding an Xpress tag at the N terminus) by site-directed mutagenesis (QuikChange Site-Directed Mutagenesis kit, Stratagene). The two genomic fragments of the AFP gene promoter cloned into the pGL3 reporter constructs, phAFP[−4995/+45]-Luc and phAFP[−178/+45]-Luc, were kindly provided by K. Riabowol (University of Calgary) and H. Nakabayashi (Hokkaido Information University). Transient transfections were performed in all experiments using FuGENE 6 (Promega), following the manufacturer’s instructions.

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For DNA affinity binding assays, biotinylated (sense) and unmodified (antisense) oligonucleotides corresponding to the AFP promoter sequence containing the ZBTB20-binding site(s) (TTCAACCTAAGGAAATACCAT AAAGTAACAGATATACCAACAAAAGGTTACTAGTT, forward strand) were synthesized (Sigma). Processing of nuclear protein extracts (NPEs) from transiently transfected cells and DNA affinity binding assays were performed as previously described36. Briefly, sense and antisense oligonucleotides were annealed in STE buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl and 2 mM EDTA). Biotinylated DNA (30 picomoles) was mixed with 200 µg of NPE in 200 µl of binding buffer (20 mM Tris-HCl, pH 7.5, 75 mM KCl, 1 mM DTT and 5 µg/ml BSA), in the presence of 10% glycerol and 6 µg of poly(dI·dC), and incubated (25 min at room temperature). Complexes were incubated with streptavidin-conjugated magnetic beads (Promega) (30 min at 4 °C and 10 min at room temperature), by mixing with rotation. Collected beads were washed three times, and bound material was eluted by boiling in sample buffer and then separated by 10% SDS-PAGE followed by immunoblotting with monoclonal antibody to Xpress (1:1,000 dilution; 46-0528, Life Technologies). Data were obtained from three independent experiments. For transactivation assays, cells were transiently transfected to express the construct(s) of interest, together with the phAFP[−4995/+45]-Luc or phAFP[−178/+45]-Luc promoter construct and 1:10 Renilla luciferase control vector (pRL-Act Renilla). In all experiments, the pRcCMV empty vector was used to equalize the amounts of transfected DNA. After transfection (24 h), cell lysates were centrifuged, and aliquots were used to determine firefly and Renilla luciferase activities with the Dual-Luciferase Reporter Assay System (Promega). Data were normalized to the activity of Renilla luciferase. Titration experiments (with wild-type ZBTB20 and the His596Arg ZBTB20 mutant) were carried out using HEK 293T cells and the phAFP[−178/+45]Luc vector, which encompasses the AFP promoter minimal responsive sequence. Transactivation assays performed to analyze the dose-dependent inhibitory activity of wild-type ZBTB20 on the phAFP[−178/+45]-Luc construct documented that maximum inhibition was attained with a 1:1 ratio (Supplementary Fig. 7). Assays using cells expressing each of the diseasecausing ZBTB20 mutants, alone or in the presence of wild-type ZBTB20, were performed using a 1:1 ratio with the selected AFP promoter construct. All assays were performed in triplicate. Confocal laser scanning microscopy. Approximately 1 × 105/ml HEK 293T cells were seeded on glass coverslips, maintained in culture in complete medium (24 h) and transiently transfected with 500 ng of constructs encoding

doi:10.1038/ng.3035

Xpress-tagged ZBTB20. Twenty-four hours after transfection, cells were either fixed with 3% paraformaldehyde (30 min at 4 °C) and permeabilized with 0.5% Triton X-100 (10 min at room temperature) or treated with CSK buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, supplemented with phosphatases and proteases inhibitors) before being fixed. Cells were stained with monoclonal antibody to Xpress (1:100 dilution) and Alexa Fluor 488–conjugated goat anti-mouse secondary antibody (1:100 dilution; Molecular Probes). After staining, coverslips were extensively rinsed and mounted onto microscope slides using Vectashield with DAPI mounting medium (Vector Laboratories). Analyses were performed in three independent experiments (>100 Xpress-positive cells observed in each experiment for each experimental condition) on a TCS SP2 AOBS apparatus (Leica Microsystems), using a 63×/1.4 N.A. oil objective and excitation spectral laser lines at 405 and 488 nm. Image acquisition and processing were performed as previously reported37. Statistical analysis. The Pearson χ2 test was used to evaluate the statistical significance (at a 95% level) of differences in proportions between experimental groups. Differences in the distribution of continuous variables between groups were evaluated for statistical significance using Student’s t testing (one-sided or two-sided distribution). In all comparisons, P values of ≤0.05 were considered to be statistically significant. 16. Dalal, P. et al. Neurology 75, 284–286 (2010). 17. Posmyk, R. et al. Am. J. Med. Genet. 155A, 2838–2840 (2011). 18. Carvalho, D.R. & Speck-Martins, C.E. Am. J. Med. Genet. 155A, 1379–1383 (2011). 19. Battisti, C. et al. J. Neurol. 249, 1466–1468 (2002). 20. Li, H. & Durbin, R. Bioinformatics 25, 1754–1760 (2009). 21. McKenna, A. et al. Genome Res. 20, 1297–1303 (2010). 22. DePristo, M.A. et al. Nat. Genet. 43, 491–498 (2011). 23. Cingolani, P. et al. Fly (Austin) 6, 80–92 (2012). 24. Liu, X. et al. Hum. Mutat. 34, E2393–E2402 (2013). 25. Schwede, T. et al. Nucleic Acids Res. 31, 3381–3385 (2003). 26. Segal, D.J. et al. J. Mol. Biol. 363, 405–421 (2006). 27. Emsley, P. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). 28. Mandell, J.G. & Barbas, C.F. Nucleic Acids Res. 34, W516–W523 (2006). 29. Hess, B. et al. J. Chem. Theory Comput. 4, 435–447 (2008). 30. Hornak, V. et al. Proteins 65, 712–725 (2006). 31. Peters, M.B. et al. J. Chem. Theory Comput. 6, 2935–2947 (2010). 32. Jorgensen, W.L. et al. J. Chem. Phys. 79, 926–935 (1983). 33. Hess, B. et al. J. Comput. Chem. 18, 1463–1472 (1997). 34. Darden, T. et al. J. Chem. Phys. 98, 10089–10092 (1993). 35. Bussi, G., Donadio, D. & Parrinello, M. J. Chem. Phys. 126, 14101 (2007). 36. Fragale, A. et al. J. Immunol. 186, 1951–1962 (2011). 37. Cordeddu, V. et al. Nat. Genet. 41, 1022–1026 (2009).

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