© 2015 Nature America, Inc. All rights reserved. tained almost 150 ( genes 150 almost tained consanguineous ( region 9q33.2-34.13 a homozygous on 11.5-Mb in single a identified mapping A) (family family autozygosity array–based SNP A A full list of author affiliations appears at the end of the paper. V) and IV types (HSAN; neuropathy autonomic and sensory CIP with for genes tive consistent were pedigrees whose autosomal recessive CIP. We excluded mutations in families the known causa two studied We therapeutics neurogenesis Prdm histone-modifying embryos. participates Prdm neural Prdm containing PRDM12 Here of therapeutic medical is In organisms Pain Tatsuo Michiue Peter De Jonghe Reinhard Windhager Maria Cilio Roberta S Samiha Shaikh Frank Reimann M Carlos Restrepo Jens Michael Hertz Luitgard Graul-Neumann Gareth T Young Andreas C Themistocleous Ya-Chun Chen pain perception Transcriptional regulator PRDM12 is essential for human Nature Ge Nature Received 29 September 2014; accepted 27 April 2015; published online 25 May 2015;

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1 48 22 was obtained from all study subjects or their legal representatives. legal their or subjects study all from obtained was consent Informed Committee. Ethics Vienna of University Medical Central, the and Committee England–Cambridge Ethics Research Medical of University Munich the East Committee NRES Service, 1 Fig. ( mutation splice-site K) obligatory an (family had individual isolated another and mutation frame-shift a an carried E) had (family subject J) isolated one (family mutation, repeat family 18-alanine one from individuals affected mutations; however, missense were variants the of majority The mutations. unrelated seven additional index patients with homozygous autosomal recessive or CIP or isolated unexplained HSAN. We found 1 Fig. ( subjects both in mutations missense identified CIP, unclassified with C genetically family and families D,family and out carried exome on sequencing from two individuals two unrelated subjects affected all ( in exon terminal alanine the in of 19 to 12 expansion from codons trinucleotide homozygous a identified and Sanger sequencing of ( B family tional candidate genes, posi these of one in mutation missense homozygous a observed we pathogenic obvious no 9, yielded chromosome on region autozygous the in A located genes in variant family from subject the of sequenc exome ing Although B. family from patient CIP unre the single and lated A family of patient index the on sequencing exome , 40 2 Fig. Fig. 1 , , Ingo Kurth & Jan Senderek , , Enza Maria Valente – 2 doi:10.1038/ng.330 42 , , Gulshan Karbani ). Subsequently, we screened screened we Subsequently, ). 36 ). This study was approved by the National Research Ethics Ethics Research National the by approved was study This ). , Bernd Rautenstrauss a and , 37 Fig. 1 Fig. 5 PRDM12 , , Alan D Irvine 10 , , Annina B Schmid Supplementary Fig. 1 Supplementary , , Adrian W Moore 5 4 , 27 17 31 , , Manuela Zitzelsberger a 49 and and , , Thomas Wieland , , , Michael S Nahorski 18 , , Roman Chrast as the only that carried different biallelic biallelic different carried that gene only the as , , Rosemarie M Watson PRDM12 8 5 Supplementary Fig. 1 Fig. Supplementary PRDM12 23 32 19 , , Diego Pereira , , Maeve A McAleer for multiple individuals from family A , ( 38 5 NM_021619. , ). ). In an approach, independent we , , Uffe Birk Jensen 6 43 PRDM12 , 11 13 , Jonathan Baets 9 , Lily , Ting-YinLily Cho , , Yesim Parman Fig. 1 Fig. , 50 Fig. 1 Fig. 8 , , Fay Stafford , 51 1 in 158 individuals with with individuals 158 in , , 5 b 2 ). We then performed performed then We ). c

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© 2015 Nature America, Inc. All rights reserved. expected forexpected recessive disease alleles ( Note ( residues and were protein predicted to interfere with normal protein function conserved strictly altered gene, the throughout distributed C-terminal a ( and tract fingers polyalanine SET zinc the three to (related domain), domain PR methyltransferase a containing acids amino 367 of ( tract. polyalanine A, motif; finger ZF, zinc domain; ( (“Shared”). individuals affected both in model”) (“AR alleles both on DB”) in (“Not databases all from excluded variants deleterious potentially containing gene circles). blue (solid D family and C family families, two ( 1–6. patients P1–P6, circle). blue (solid B family in mutation homozygous candidate region, candidate the in gene one results, inconclusive yielded circle) blue (open A family from patient index the of sequencing exome Although ideogram). 9 chromosome the to next bar vertical red a and markers SNP flanking of numbers (rs) cluster SNP reference by (represented 9q33.2-34.13 chromosome on region candidate single a pinpointed circles) red (solid individuals four of mapping autozygosity SNP-based A, family in 1 Figure s r e t t e l  c b Supplementary Fig. 2 Supplementary b a ) Schematic representation of the PRDM12 protein and distribution of mutations. Amino acid numbering is shown along the bottom. PR/SET, PR PR PR/SET, bottom. the along shown is numbering acid Amino mutations. of distribution and protein PRDM12 the of representation Schematic ) ) Exome sequencing of subjects from from subjects of sequencing Exome ) P14 P14 PRDM12 Myelinated fibers (mm–2) PRDM12 ). ). In all 11 families studied, the 1,000 1,500 2,000 500

0 Identification of mutations mutations of Identification P6 . ( . is a five-exon gene encoding a single protein isoform isoform protein single a encoding gene five-exon a is 1 PRDM12 a ) In Pakistani multiplex multiplex Pakistani In ) 3 2 Diameter ofmyelinated fibers 5 4 Healthy control P10 P6 i. 1 Fig. PRDM12 was the only only the was P7 P7 , , 7 6 Supplementary TableSupplementary 2 c ). The observed point mutations were were mutations point observed The ). 9 8 , harbored a harbored , P10 Healthy control 10

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Ala354_Ala359dup Family Italy P6 A (top two rows) or 20 20 or rows) two (top 50 bars, Scale density. a reduced at but fibers, VIP-immunoreactive by innervated were glands sweat and absent, almost were fibers nerve CGRP-immunoreactive dermal P11, from biopsy the biopsy. In subject’s affected the in line) dashed (red border dermal-epidermal the cross not did fibers nerve donor, a healthy from biopsy the in observed were arrowheads) (red endings nerve intraepidermal ample Although fibers). nerve autonomic for (VIP, peptide a marker intestinal vasoactive and afferents) primary nociceptive of subpopulation a (CGRP, peptide labeling gene-related calcitonin marker), (pan-neuronal PGP9.5 20 bars, scale blue; toluidine with stained were sections Semithin control). (healthy 9,609 and (P10) 4,438 (P6), 4,692 were millimeter square per fibers myelinated of numbers total The axons. myelinated small-caliber of loss selective ( obtained. was individuals the of images publish to Consent ulcers. foot diabetes-like and scratching facial as such sequelae with phenotype a milder represented J) (family P18 and P17 Patients phalanges. distal of mutilation and scarring opacity, corneal lips, and tongue with 2 Figure general the in polyalanine PRDM12 of (refs. rs942153 rs578802

b Ala353_Ala359dup ) Sural nerve biopsy specimens showing showing specimens biopsy nerve ) Sural B PRDM12 q p Chr9 16

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© 2015 Nature America, Inc. All rights reserved. consisted of facial scratching, diabetes-like foot ulcers, intact corneal corneal intact foot ulcers, diabetes-like scratching, of consisted facial normal. were and families other the hearing in than milder and was phenotype J, the family smell In of senses the observed; was tion No autonomic dysfunc other members. family to unaffected respect with reduced substantially were but occurred tearing and Sweating normal. mostly were proprioception) and vibration touch, (light ties modali sensory large-fiber Notably, life. in later joints neuropathic and to deformities led had bone and of joints bones and occasionally of skin the infections recurrent individuals, affected In severely ring. scar corneal progressive to led which absent, were reflexes Corneal ( as burns and well episodes as traumatic unnoticed self-biting, repeated from to resulting due injuries fingers and tissues the perioral of tongue, lesions mutilating painless numerous these sustained children individuals and infants as Consequently, cold. or heat identify not noxious could and birth from pain inflammatory or acute feel PRDM12 exceptional. are J family and A family in and ( alanines 14 of maximum a with polymorphic, is population Nature Ge Nature were carriers Heterozygote tearing. and sweating intact and reflexes MO–treated embryos are indicated. *** indicated. are embryos MO–treated Prdm12 and MO–treated control in expression between differences Statistical condition). per embryos ref. from modified stage; tailbud late in placodes 200 bars, Scale placode. trigeminal arrowheads, whole-mount by 28) (stage Islet1 ( development placode sensory cranial of markers for staining irregular caused embryos ( process. differentiation of day D, neurons. sensory of differentiation the during development of stages the illustrates map heat the above drawing schematic The differentiation. successful confirmed markers neuron sensory canonical and markers pluripotency of expression the in Changes specification. crest neural during that showed neurons sensory iPSC-derived human of ( (P56). DRG mature in and (E9.5–P14) differentiation neuron sensory and development DRG of period Prdm12 100 250 left, bars: Scale arrowheads). (black DRG in strong showed E10.5 at (right) cord spinal cervical of sections transverse and (middle) embryos whole of arrowhead). (white migration and delamination cell crest neural of stage earliest the with coincided which Prdm12 of expression identified (left) E9.0 at situ in ( development. neuron 3 Figure a E9.0 The phenotype of the affected individuals in the 11 families with with families 11 the in individuals affected the of phenotype The Supplementary Fig. 3 Fig. Supplementary µ ). Embryos injected with control MO or Prdm12 MO were analyzed at the late tailbud stage stage tailbud late the at analyzed were MO Prdm12 or MO control with injected Embryos ). m. ( m. hybridization of mouse embryos embryos mouse of hybridization Fig. 2 Fig. expression throughout the whole whole the throughout expression arrowhead), (black folds neural in

A role for Prdm12 in sensory sensory in Prdm12 for A role µ mutations was largely consistent: they had been unable to to unable been had they consistent: largely was mutations b m; middle, 500 500 middle, m; ) RT-PCR analysis confirmed confirmed ) RT-PCR analysis Xenopus laevis Xenopus n PRDM12 etics a , , E10.5 Supplementary Note Supplementary In situ In

c Prdm12 expression peaked peaked expression ) Quantitative RT-PCR ) Quantitative ADVANCE ONLINE PUBLICATION ONLINE ADVANCE a ) Whole-mount ) Whole-mount hybridization hybridization are shown in the schematic drawing at the top of the panel (lateral view, (lateral panel the of top the at drawing schematic the in shown are µ in situ in m; right, right, m; ). This confirms that the alleles observed observed alleles the that confirms This ). expression expression E10.5 hybridization; yellow arrowheads, profundal placode; green green placode; profundal arrowheads, yellow hybridization; d ) Knockdown of Prdm12 by a specific morpholino (MO) in in (MO) morpholino a specific by Prdm12 of ) Knockdown 2

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D7 tube E10.5 D6 U E11 that are destined to become nociceptors. Therefore, we explored explored we Therefore, nociceptors. become to destined are that in morphologically ( were normal grossly but individuals unaffected in observed was what to compared reduced were glands sweat of innervation nomic thermoceptors and nociceptors of terminals the the representing innervate (normally to epidermis membrane basement the crossing fibers nerve of absence a complete We affected. observed were of C fibers terminals skin biopsies of two CIP patients that suggested at least the peripheral (ref. available were microscopy electron for samples suitable no as biopsies, nerve qualitative changes in C could fibers not be reliably in determined the other were ( largely unaltered for modalities sensory axons large-caliber whereas patients, two of nerves sural nociceptors of 70% 30% of nociceptors, and unmyelinated C fibers, normally constituting A myelinated (small neurons sensory nociceptive of projections peripheral evaluate to study, our before years several purposes diagnostic for done patients, CIP of biopsies We perception. pain nerve and had normal studied asymptomatic all -test). -test). D9 Neural crestcell E11.5 specification Our clinical and histological findings suggested that mutations mutations that suggested findings histological and clinical Our PRDM12 crest cells D11

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8 P14 Sensory neurons Fig. 2 Fig. ), whereas the subepidermal neural plexus and plexus neural auto ), the subepidermal whereas D12 and and

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Control MO 20 40 60 80 0 Olfactory Lens Control MO Prdm12 MO 50 δ Ath3 fibers, normally constituting constituting normally fibers, Mildly abnormal ** 46 Fig. Fig. 2

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* Control MO * δ Severe defects Lateral line fibers in the the in fibers Prdm12 MO 53 Islet1 53 ** n  - © 2015 Nature America, Inc. All rights reserved. onic stem cells also showed robust induction of induction robust showed also cells embry stem onic human from derived neurons nociceptor-like addition, In ( current sodium tetrodotoxin-resistant neuronal maturation recording of and confirmed nociceptor-specific after cells these of properties electrophysiological the Weexamined by then increased more and at than 1,000-fold peaked day 9 ( Expression specification. crest neural with commensurate 7 day on neurons nociceptor-like humans, we differentiated inducible pluripotent stem cells (iPSCs) into assess To( DRG the in cells Schwann or cells glial Prdm12 ( 14) day (E14.5–postnatal differentiate and mature (E10.5–E13.5), in not emerge neurons but sensory when (DRG)) time the during ganglia ganglia sympathetic root (dorsal ganglia spinal sensory in bodies cell nociceptor contain that ganglia sensory the including tissues, various into develops that population cell migratory multipotent, transient, a of consists crest ( cells crest neural to rise give which folds, ral ( of expression mice, In cells. stem from generated neurons of expression the *** * significant. not ns, indicated. are mutants Prdm12 and type) (wild control between differences Statistical s.d. represent bars in graphs bar The domain. PR the of structure the affect may core hydrophobic the into (p.Trp160Cys) partner a disulfide-bond or (p.Ile102Asn) chain side a polar of Introduction domain. PRDM12-PR the of core the in (cyan) residues other with interactions hydrophobic have Trp160 and (orange) Ile102 residues ( G. immunoglobulin IgG, fingers. zinc lacking mutant artificial an is Prdm12 ion. zinc the coordinate that (cyan) residues the of one is (orange) His289 domains, finger zinc PRDM12 the for model structural the to According lysate. the in protein G9a and fraction (IP) immunoprecipitation the in Prdm12 of amounts to normalized was G9a Bound Myc). (IP: anti-Myc using immunoprecipitated and (Input) cells COS-7 in expressed were FLAG-G9a and Myc-Prdm12 interaction. Prdm12-G9a impairs alteration ( H3. total and (Myc) Prdm12 to normalized were signals H3K9me2 15). (stage stage mid-neurula until cultured mutant), and type Myc- with microinjected were functionless. were mutants missense CIP-associated but neurula, in H3K9 on dimethylation robust ( staining). (DAPI nuclei blue, tag); anti-HA the of detection (fluorescent PRDM12 Red, (bottom). cells HEK-293T in cytoplasm and nucleus the in aggregates formed mutant transfected The control). (transfection-efficiency of those to normalized were signals HA-PRDM12 MG132. by recovered was expression PRDM12; wild-type did than cells COS-7 in levels expression lower showed mutant expansion polyalanine ( 4 Figure s r e t t e l  ( differentiation during a NM_00112336 Fig. 3 Fig. ) The hemagglutinin (HA)-tagged PRDM12 PRDM12 (HA)-tagged hemagglutinin ) The P < 0.001 (Welch’s < 0.001 b

was expressed primarily by neurons rather than by satellite satellite by than rather neurons by primarily expressed was and and Consequences of of Consequences PRDM12 Supplementary Fig. 5a Fig. Supplementary 2 b Wnt8 ) starts ) around day starts embryonic 9.0 in (E9.0) the neu ) Wild-type Prdm12 induced induced Prdm12 ) Wild-type Xenopus Prdm12 c expression during nociceptor development in in development nociceptor during expression ) The p.His289Leu p.His289Leu ) The α t and and -tubulin and GFP GFP and -tubulin -test). d 22 Supplementary Fig. 6c Fig. Supplementary ) Mutation-altered ) Mutation-altered PRDM12 animal cap cells cells cap animal during embryogenesis and in human pain pain human in and embryogenesis during , Chrd 2 Prdm12 3 . . PRDM12 Xenopus mRNA and and mRNA 2 0 . . mutations. mutations. ∆ Prdm12

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renders the protein less stable and more susceptible to proteolysis, proteolysis, to susceptible more and stable less protein the and renders PRDM12 of inhibition, aggregation causes proteasome expansion the that upon suggesting recovered were mutant expansion ( nucleus and cytoplasm in PRDM12 foci to and cells concentrated discrete, form caused in transfected the overexpressed of levels reduced in resulted mutation localization subcellular nor ( expression protein neither affected mutations missense that Wefound pattern. lace-like diffuse, a protein with nuclear a is PRDM12 function. protein PRDM12 for tions neurogenesis sensory of regulator universal vertebrates. in a is Prdm12 that ( normal mostly seemed placodes 28; (stage stage tailbud late the at placodes sensory cranial of genes marker of distribution irregular of ( study ortholog frog the of a Knockdown by supported further zebrafish and mice of cords spinal in and brain mouse in observed been also has of expression limited that note we (although neurogenesis nociceptor during Prdm12 of function essential an with consistent DRG ( the find not did A353_A359dup Supplementary Fig. 9a Fig. Supplementary 10

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C273 Glu172Asp FLAG Myc Myc-Prdm12 FLAG-G9a Myc-Prdm12 His289Leu Myc-Prdm12 wildtype N Nature Ge Nature IgG heavychain 1 Zn His289Leu ) resulted in an an in resulted ) 2+ C276 morphants. morphants. H289 ), suggesting ), suggesting P H293 < 0.01, < 0.01, ∆ ZF H3 H3K9me2 Myc Prdm12 n etics C 2 5 -

© 2015 Nature America, Inc. All rights reserved. R.C. andR.C. J.S.), and Friedrich-Baur Stiftung (J.S.). 1140 to C.B. and toKU1587/4-1 I. Kurth), Stiftung Gebert-Rüf to(GRS-046/09 (NEUROMICS) to J.B. and P.D.J.), Deutsche Forschungsgemeinschaft (CRC/SFB contre les Maladies Neuromusculaires and EU (grantFP7/2007-2013 2012-305121 M.A.-G.), the UK Medical Research Council (M.S.N. and S.S.S.), Association Belge Research Centre (Y.-C.C., F.S. and C.G.W.), Austrian FondScience to(P23223-B19 (ref. no. This 095698z/11/z). work was supported by Cambridge NIHR Biomedical helpful suggestions on the text. D.L.H.B. is a senior Wellcome Scientist Clinical A, J.R.P. Madrid and F. Axelrod for advice and discussion and M.F. Passarge for staff is greatly appreciated. We thank S. Malik for her invaluable work with family in this study. The help of all contributing medical, technical and administrative The authors are grateful for the participation of the patients and their families online version of the pape Note: Any Supplementary Information and Source Data files are available in the the the of in version available are references associated any and Methods Met through relief pain nociceptors. overactive of the of reprogramming methods new for target possible a suggests circuits nociceptive cord spinal and peripheral to the in found changes epigenetic being with are correlate pain neuropathic and chronic of mechanisms genesis, nociceptor via G9a possibly or in related factors points critical during modification histone of control of loss a involves mechanism pathological the that imply data Our perception. of pain loss a congenital cause mutations genic protein-binding the alter PRDM12-PR. of capability may residues these affecting mutations PR the of modules As protein-interaction represent domain. PR domains surface the to contribute Glu172 and Arg168 structure. ( domain PR the of core the ( 9 determined Fig. be with to interfere remains mutants dimethylation Prdm12 H3K9 other which by mechanism The the of ( finger ion zinc second zinc the coordinating residues the of one is His289 that 9c Fig. Supplementary to G9a, whereas the other mutants bound G9a normally ( normally G9a bound mutants other the whereas to G9a, binding reduced significantly alteration p.His289Leu the that found we Mechanistically, capacity. methylation histone impairing by CIP that hypothesis the supports by differed ~15% of protein amino their acids ( frog the and orthologs mammalian the although in embryos, H3K9me2 induced strongly Prdm12 wild-type mouse ( effect an such demonstrate not did stage embryos, whereas the CIP-associated missense Prdm12 mutants in H3K9me2 Prdm12 robustly increased wild-type of effect diseases human other in netic mechanisms cause defects in neuronal development, as observed checkpoints epigenetic during neurogenesis (H3K9me2) the recruits and H3 histone at to 9 G9a dimethylate (Ehmt2) lysine methyltransferase activity methyltransferase histone intrinsic lacks neurogenesis in the control of that participate vertebrate regulators transcriptional alanine expansions in humans poly nucleus. pathogenic other for the reported been have within observations Similar availability biological its reducing eventually Nature Ge Nature A cknowledgments PRDM12 is essential for the sensing of pain in humans, as patho as humans, in pain of sensing the for essential is PRDM12 h ). Substitutions of Ile102 and Trp160, which are located in in located are which Trp160, and Ile102 of Substitutions ). o PRDM12 d s n 3 etics 0 7 pape , . Histone modifications have emerged as critical critical as emerged have modifications Histone . 8 , 24– mutations on H3K9me2 levels. Overexpression of Overexpression levels. H3K9me2 on mutations

2 r 9 Fig. 4 Fig. 9 . r ADVANCE ONLINE PUBLICATION ONLINE ADVANCE , . Unlike other Prdm family members, Prdm12 Prdm12 members, family Prdm other Unlike . . 1 0 . The histone-modifying activity of PRDM12 PRDM12 of activity histone-modifying The . ). The putative structure of PRDM12 implies implies of PRDM12 structure putative The ). 35– c ), which is required for G9a association for G9a required is which ), 3 Fig. 4 Fig. 8 16 . Therefore, we next investigated the the investigated next we Therefore, . PRDM12 , 1 7 3 . . Prdm12 is a member of a family of 4 d . Increasingly, pathophysiological . Increasingly, pathophysiological ), are likely to alter the domain’s the alter to likely are ), Fig. 4 Fig. Supplementary Fig. 10 Supplementary missense mutations cause cause mutations missense b 31– ). Also, both human and and human both Also, ). 3 4 , , and aberrant epige Xenopus Supplementary Supplementary

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18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 3. 2. 1. reprints/index.ht at online available is information permissions and Reprints The authors declare no competing interests.financial other authors. The manuscript was written by Y.-C.C., M.A.-G., C.G.W. and J.S. with input from the project, participated in data analysis and the directed andresearch. supervised J.W., I. Kurth and D.L.H.B. gave advice. critical M.A.-G., C.G.W. and J.S. oversaw pluripotent stem cells. andR.S. J.S. outcarried protein modeling. A.W.M., R.W., embryos. Y.-C.C., L.T.-Y.C. and G.T.Y. were responsible for experiments involving biopsies from CIP patients. S.M. and T.M. performed experiments in mutations in M.D.,R.S., C. Stendel, F.R., T.M. and J.S. consequences functional assessed of andR.S. performed expressionR.C. studies on analysis and M.S., T.W., F.S., M.S.N., S.S.S., O.P.C., A.K.N., C.G.W. and J.S. outcarried linkage samples, skin biopsies and biopsynerve specimens. Y.-C.C., M.A.-G., T.M.S., C.W., C.B., B.R., J.B., P.D.J., I. R.K., M.M.R., Kurth, C.G.W. and J.S. obtained DNA study and provided patient care. Y.P., L.G.-N., E.P., J.M.H., E.M.V., P.J.W., M.R.C., G.K., M.A.M., J.C.M., S.M.M., A.D.I., U.B.J. and C.G.W. enrolled patients in the M.A.-G., Y.P., L.G.-N., W.H., R.M.W., J.M.H., U.M., M.B., D.P., K.v.A.,C.M.R., C.F., 24. 23. 22. 21. 20. 19. 6. 5. 4. COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS

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© 2015 Nature America, Inc. All rights reserved. should should be addressed to C.G.W. ( Sweden. Bochum, Germany. Diseases, Neuromuscular UCL Institute of Neurology, National Hospital for Neurology, London, UK. Institute Born-Bunge, University of Antwerp, Antwerp, Belgium. Munich, Germany. Medical Center, Freiburg, Germany. University Hospital, Aarhus, Denmark. Ireland. 35 of California San Francisco, San Francisco, California, USA. (GENetic DIAgnostic Network), Antwerp, Belgium. Germany. Munich, Germany. del Rosario, Bogotá, Colombia. San Giovanni Rotondo, Italy. Germany. Ireland. Leipzig, Germany. Berlin, Berlin, Universitätsmedizin Germany. St. Lucia, Australia. Institute, Saitama, Japan. Munich, Germany. South Africa. of Oxford, Oxford, UK. 3 1 Tokyo, Japan. 33. 32. 31. 30. 29. 28. 27. 26. 25. s r e t t e l  Department Department of Medical Orthopaedics, University Vienna, Vienna, Austria. Department of Medical Genetics, University of Cambridge, Cambridge, UK. Department Department of Neurology and Neurophysiology, Our Lady’s Children’s Hospital, Dublin, Ireland.

oe EM, cut, .. Za, . rstl aog pgntc pathways epigenetic among Crosstalk X. Zhao, & A.L. McQuate, E.M., Jobe, a, S.L. Tan, stem neural in choice fate cell Wang,on Y.X.L., control Hu, Epigenetic Q. Shen, & anti- exhibits and acid retinoic by induced is Prdm12 Y. Shinkai, & C.M. Yang, Chittka, A., Nitarska, J., Grazini, U. & Richardson, W.D. Transcription factor positive Endo, K. T.Kaji, C.C., Rossi, Artinger,& TranscriptionalK.B. sensory Rohon-Beard of control single- between switch genetic A.W.,L.Y.Y.N.binary Moore, Jan, a &Hamlet, placodes. cranial of specification and Induction G. Schlosser, regulates neurogenesis. regulates (2012). 3806–3816 epigenetic control of neural progenitor cells during development. cells. embryonic P19 of modulation cycle cell cells. carcinoma the through properties proliferative differentiation. and proliferation cell stem neural control and methylation arginine histone mediate to regulatory domain 4 (PRDM4) recruits protein arginine methyltransferase 5 (PRMT5) diversification. border. plate neural the (2009). at development neuron morphology. neuron dendrite multiple- and (2006). 303–351 37 20 51 Protein Cell Protein 30 22 Department Department of Clinical Genetics, Odense University Hospital, Odense, Denmark. Academic Academic Unit of Neurology, Trinity College, Dublin, Ireland. Department Department of Clinical Karolinska Neuroscience, Institutet, Stockholm, Sweden. CharitéCentrum für CharitéCentrum Zahn-, Mund- Berlin, Berlin, Universitätsmedizin und Germany.Kinderzahnmedizin, Arbeitsbereich Kieferheilkunde, Ambulanz, Krankenhaus Neuropädiatrische der Barmherzigen Schwestern Linz, Linz, Austria. et al. 8 5 t al. et Institute Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany. Friedrich-Baur-Institute, Friedrich-Baur-Institute, Ludwig Maximilians University Munich, Munich, Germany. Chromatin modification of Notch targets in olfactory receptor neuron Nat. Neurosci. Nat. 18 28 10 44 seta rls f h hsoe ehlrnfrs EE i the in ESET methyltransferase histone the of roles Essential 49 Cell Struct. Funct. Struct. Cell 14 Institut Institut für Essen, Essen, Universitätsklinikum Humangenetik, Germany.

Department Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK. Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland. Neurogenetics Group, Neurogenetics VIB Department of Molecular Genetics, University of Antwerp, Antwerp, Belgium. 3 Institute Institute of Human Genetics, Jena University Hospital, Jena, Germany. Department of Neurology, Istanbul University, Istanbul, Turkey. J. Biol. Chem. Biol. J. , 278–290 (2012). 278–290 , 7 Brain Brain Function Research Group, School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 12 Front. Neurosci. Front. Neusentis Neusentis Research Unit, Pfizer, Cambridge, UK. 24 Departamento Departamento de Cirugía Plástica, Hospital Infantil de Universitario San José, Bogotá, Colombia. 26 [email protected]

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e. Biol. Dev. Department Department of Neurology, Antwerp University Hospital, Antwerp, Belgium. 238 931–943 , 38 Clinical Clinical Medicine, Trinity College, Dublin, Ireland. 4 Department Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo,

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m J. Am. © 2015 Nature America, Inc. All rights reserved. and 156 (P8), with 91% (P3), 89% (P6), 95% (P7) and 97% (P8) of the the of (P8) 97% and (P7) 95% (P6), 89% (P3), 91% with (P8), (P7) 156 102 (P6), and 93 (P3), 94 were depths read average The Gb. (P8) (P7) 12.3 8.2 and (P6), 6.2 (P3), 6.4 of sequences in resulted which runs, paired-end 100-bp Weperformed (Agilent). kit Mb 50 Exon All Human SureSelect the For of (Illumina). enrichment we exons intronic used and sequences, flanking P7 C) (family and P8 D) (family using a 2000 system HiSeq Genome Analyzer ofsequencing the index patient of family A (P3) and individuals P6 (family B), sequencing. Whole-exome individuals. four with analyzed were EXCLUDEAR calls allele the then and (Affymetrix), chips SNP Nsp1 250K using processed were samples DNA Genomic A. family from viduals studies. Linkage artifacts. fixation and vessels blood spaces, perineural avoid to taken was care analysis, for areas of selection the In ally. manu traced was fiber of intact each sheath myelin and the by observers, the sies were used for Myelinated evaluation. morphometric were fibers identified biop of nerve cross-sections semithin of blue–stained Micrographs toluidine at (P6, biopsied from biopsies two age subjects 25, and at P10, biopsied age 7). of images the microscopic reevaluate before to able years Wewere place. took several study current biopsies nerve sural diagnostic had had P11) and biopsies. nerve Sural magnification. 63× and at 40× Zeiss) (Carl objective Plan-Apochromat a with microscope 700 an LSM using taken were Images IgG. anti-mouse key Cy3-conjugated donkey anti-rabbit IgG and Alexa Fluor 488–conjugated don mouse anti–myelin basic protein (clone SMI-94, Abcam, ab24567) followed by myelinated fibers in the lip skin, we stained sections with rabbit anti-PGP9.5 and 488–conjugated donkey anti-mouse IgG (Life Technologies, A-21202). To assess Fluor Alexa with sc-7841) IgG ( Santa Cruz, clone and M-19, anti-VIP mouse were detected using rabbit anti-PGP9.5 with Cy3-conjugated peptide donkey anti-rabbit of intestinal autonomic (a effectors) subpopulation fibers nerve (VIP)-immunoreactive Vasoactive 713-165-003-JIR). (Stratech, IgG anti-sheep sheep and IgG donkey Cy3-conjugated with BML-CA1137) anti-rabbit Sciences, Life (Enzo anti-CGRP goat IgG 488–conjugated Fluor Alexa anti-rabbit with PGP9.5 anti- (CGRP)- rabbit with goat co-staining peptide by assessed were gene-related nociceptors 488–conjugated immunoreactive Calcitonin Fluor A-11034). Technologies, Alexa (Life or ImmunoResearch, (Jackson 711-165-152) IgG anti-rabbit donkey Cy3-conjugated with described as microscopy immunofluorescence for processed 50- into cut nitrogen, liquid in frozen Finetek), °C. 4 at h 24 for (Sakura Tissue-TekO.C.T. in Compound buffer embedded then were samples phosphate Skin with sucrose 10% in stored and buffer was in fixed specimen Zamboni’s solution for 4 h at 4 °C, washed in phosphate tissue The injuries. perioral for surgery during 13) age (at from P7 of lip lower the obtained was biopsy skin second A °C. 4 at h 48 for buffer phosphate with sucrose 15% in stored and buffer phosphate in washed °C, 4 at h 24 for periodate-lysine-paraformaldehyde 2% in fixed was sample The 18). age (at biopsies. Skin in listed in RT-PCRfor and listed are are study this in 5 Table used Supplementary antibodies Primary indicated. otherwise Materials. production. tear for test Schirmer a had (P3) one and test flare axonal histamine a had P12) and (P3 two test, a sweat had P11) and (P7 two examinations, neurophysiological underwent P16–P21) and P13 P11, P10, P8, (P7, patients Eleven sequencing. clini and HSAN CIP similar for cally genes causative known the in Mutations authors. the of studies. Clinical O doi: performed was alignment Read 20-fold. least at covered regions targeted NLIN Nerve fibers were visualized using rabbit anti-PGP9.5 (Ultraclone, RA 95101) 10.1038/ng.3308 E E Specific reagents were purchased from Sigma-Aldrich unless unless Sigma-Aldrich from purchased were reagents Specific M ET 4 A punch skin biopsy was obtained from the lower leg of P11 P11 of leg lower the from obtained was biopsy skin punch A 4 . Only one homozygous region >1.5 cM was shared by all all by shared was cM >1.5 region homozygous one Only . H Patients were in after seen being the recruited by clinic some uoyoiy apn ws efre o fu indi four on performed was mapping Autozygosity O 11– D Four individuals from different families (P3, P6, P10 P10 P6, (P3, families different from individuals Four 1 5 S ( . Primers used for generating expression constructs constructs expression generating for used Primers . Supplementary Table 4 Supplementary We carried out targeted exome capture and and capture exome targeted out carried We Supplementary Table 6 Supplementary µ m free-floating sections and sections m free-floating ) were ruled out by Sanger Sanger by out ruled were ) . 4 3 . - - - - -

BLAST with default settings against the NCBI nonredundant database. We database. nonredundant NCBI the against settings default with BLAST ( PRDM12 human to homologs for ( sequences PRDM12 on substitutions acid (ref. analysis. Protein-sequence Biosystems). (Applied DNA Analyzer 3730 PRISM ABI an on analyzed and Biosystems) (Applied Kit Sequencing Cycle Reaction Ready Terminator BigDye PRISM ABI the using determined were products PCR of Sequences plates (Macherey-Nagel). PCR NucleoFast using clean-up purified were Amplicons request. upon available are conditions PCR and sequences the and assembly) the of basis the on genomic sequence designed of primers using amplified were sites splice and families. additional in detection Mutation variants. Only genes containing variants werebiallelic considered further. were annotated with custom scripts, and Variantswe P8). retained (P7, onlydiseases unrelated potentially with deleteriousindividuals from exomes in-house the 1000 Genomes data (P3, P6) or for variants present in more than 8 of >4,000 any of 100 in-house controls (exomes from individuals with other diseases) average or in average an heterozygosity of >0.02 (P7, P8). We next filtered for variants with found in dbSNP132 with in heterozygosity called present of >0.02 (P3, P6) were or variants HapMap SNPs present deletions in excluded dbSNP135 with an and We SAMtools. insertions small and variants nucleotide with BWA (version 0.5.8) to the assembly GRCh37/hg19. Single- (developmental stages E12 and beyond), and whole embryos and neural neural and embryos whole and possible beyond), when and isolated E12 were stages (DRG) (developmental ganglia root Dorsal mice. C57BL/6J mouse of analysis RT-PCR Prdm12 from synthesized pGEM-T Easy linearized plasmid vector containing a partial phosphate previously described as Roche) (NBT/BCIP; tetrazolium/5-bromo-4-chloro-3-indolyl chromophoric blue the with nitro detection substrate signal and probes digoxigenin-labeled mRNA section and whole-mount for processed situ In into subcloned and PCR Matsukawa (S. elsewhere described preparation). Mutants of is vector pCS2 in Prdm12 Myc-tagged for construct expression an of Generation embryos. 22 (Prdm12 pCMV-Myc. into motifs finger zinc subcloned and Japan) Saitama, three Institute, by Yoichi (RIKEN provided Shinkai all lacking mutant Prdm12 Eco into inserted were sequences Mutated PCR. overlap-extension using erated gen were Mutations pCMV-Myc. vector expression mammalian the of sites into inserted and embryos whole E13.5 mouse from mRNA HA tag. N-terminal an with vector pCDNA3 the into introduced were ucts prod The full-length as template. the fragments both with PCR recombinant and 5 PRDM12 The remaining coding sequence of GCGGCTTCAACTCGCGCA-3 5 primers the using DNA genomic patients’ from amplified was expansion PCR overlap-extension by mutations obtained were expansion polyalanine The (Agilent). Kit Mutagenesis commercially Directed tagging Site- II QuikChange the using by introduced were mutations Point HA tag. epitope vector N-terminal an with pcDNA3 BioScience) (Source a template PRDM12 in purchased PRDM12 human type constructs. expression of Cloning ( settings default with ClustalW using alignment multiple-sequence a created Supplementary Fig. 2 Fig. Supplementary Xenopus laevis Prdm12 laevis Xenopus mouse Myc-tagged RI and and RI 4 ′ 7 -TGCGCGAGTTGAAGCCGC-3 hybridization of mouse embryos. mouse of hybridization ), PolyPhen-2 (ref. (ref. ), PolyPhen-2 mRNA construct using the primers 5 primers the using construct Not I sites of pCMV-Myc. An expression plasmid for an artificial artificial an for plasmid expression An pCMV-Myc. of sites I 8 . PRDM12 PRDM12 Prdm12 ). Xenopus Eco cDNA ( 4 8 WePROVEAN used ) ) and SNPdryad RI and and RI Prdm12 mRNA mRNA (chr9: 133,539,981–133,558,384, GRCh37/hg19 cDNA ( cDNA ′ Prdm12 were obtained by overlap-extension and 5 and NM_00107943 PRDM12 upeetr Tbe 2 Table Supplementary Xba e eeae te osrc fr wild- for construct the generated We . Tissues were collected from wild-type wild-type from collected were Tissues NM_021619. NM_00112336 ′ ′ I sites of the pCS2 vector. pCS2 the of sites I . Gel-purified products were . used for Gel-purified -TCACAGCACCATGGCCGGC-3 ′ -ATGATGGGCTCCGTGCTCC-3 5 Mouse embryos (C57BL/6J) were were (C57BL/6J) embryos Mouse 0 was amplified from the wild-type . . 4 8 PRDM12 9 , The The to analyze the effects of amino effects the to analyze 5 NP_06763 1 . The The . 0 4 5 in situ in ) was obtained from stage- from ) obtained was , SIFT , 2 PRDM12 sequence. The primer primer The sequence. Prdm12 2 exon 5 harboring the the harboring 5 exon ) was generated from from generated was ) Nature Ge Nature hybridization with with hybridization 4 2 6 ) by running PSI- running by ) , MutationTaster2 , ). We gathered gathered We ). Eco coding region region coding riboprobe was was riboprobe RI and and RI ∆ t al. et ZF) was was ZF) Xenopus n etics Not , in in , ′ ′ - - - I ′ .

© 2015 Nature America, Inc. All rights reserved. solutions containing 140 mM KCl, 1.6 mM MgCl mM 1.6 KCl, mM 140 containing solutions and 5 the mM pH glucose; was set at with 7.4. intracellular Pipettes were filled taining 140 mM NaCl, 4 mM KCl, 1.8 mM CaCl con solution extracellular in recorded were channels sodium Voltage-gated 80%. and 70% between compensated was resistance series and compensated, M 2–4 of with used were Products) (Science pipettes borosilicate Glass Devices). (Molecular 2000 Digidata a by digitized and Devices) (Molecular 700A amplifier Multiclamp a using made were recordings Patch-clamp dissociation. sensory iPSC-derived d of cellular 1–2 within factor) differentiated of growth addition the after weeks (6 neurons fully on neurons. performed sensory were recordings iPSC-derived of recordings Electrophysiological Technologies). (Life packages Bioconductor to normalized that of PRISM 7900HT ABI machine an (Applied on Biosystems). Gene Technologies) expression was (Life analyzed, sets probe TILDA primer Taqman containing customized cards and Biosystems) (Applied mastermix expression gene kit (Applied Quantitative RT-PCRBiosystems). using was Taqmanperformed neurons. sensory RNA-to-cDNA Then cDNAusing a treatment. high-capacity was synthesized iPSC-derived DNaseI on-column with Kit Mini and RNeasy the using isolated was Total RNA hESC- of RT-PCR Quantitative population. non-neuronal the reduce to C mitomycin with treated ng/ml Cultures were 25 culture. and for was added used long-term concentration) final at used and Peprotech from all GDNF, human and NT-3 with BDNF, NGF-b (N2B27 medium maturation 11, day On N2B27 10). (100% day 4 on day on medium starting day other every increments 25% in added ml 3.5 Technologies), ml 5 Life from supplement, (all B-27 ml L-glutamine 10 supplement, N-2 ml 5 medium, ml (480 neurobasal medium N2B27 and daily, fed were Cells phenotypes. crest neural CHIR99021 3, day From (3 specification. neuroectoderm anterior drive to (1 ml nones 3.5 Technologies), ml β Life from 5 (all L-glutamine Replacement, ml 5 acids, Serum amino sential Knockout ml 100 DMEM, Knockout Matrigel on medium ml (390 medium KSR 0). (day Technologies) induction neural h 48 before Biosciences) (BD (StemCell mTeSR1 in cells single as reported previously as performed was differentiation ments, but they were not further authenticated. hESC and iPSC sensory neuron experi for used being before free mycoplasma be to confirmed RT-PCR and quantitative by markers pluripotency express to shown were lines Cell UK). Cambridge, (Pfizer, Unit Research Neusentis by generated was F Sendai line (New York, Pfizer from and iPSC H9 the obtained was line USA), (hESC) cell hESC and neuron iPSC differentiation. sensory were bands light. UV under DNA visualized and gels, agarose on examined and run were Amplicons PCR was using performed 100 ng of cDNA and Hot Star Master Mix (Qiagen). Clontech. from purchased were II) and I Panels MTC (Human panels cDNA PRDM12 light. UV under imaged Technologies). Amplicons were resolved on agarose gels, and DNA bands were G2 GoTaq either and (Life or TaqPolymerase DNAPlatinum (Promega) Polymerase primers High-Fidelity of variety a used we PCR, For (Qiagen). Kit RT Omniscript or (Takara Bio) Kit RT Reagent PrimeScript the with formed per was transcription Reverse (Qiagen). Kit Mini RNeasy the using cultures previously described as Egr2 markers cell Schwann of detection by determined was cells the of Identity mycoplasma. for negative tested and passages low at maintained from Professor Roman Karolinska Chrast, Institutet, Sweden) was Stockholm, gift (a MSC80 line cell Schwann mouse The embryos. were E13.5 from ganglia dissected Sympathetic stages. developmental earlier at used were tubes Nature Ge Nature We elicited 7.3. at set was pH the HEPES; mM 10 and EGTA NaGTP,M 2m -mercaptoethanol) containing small-molecule inhibitors LDN193189 LDN193189 inhibitors small-molecule containing -mercaptoethanol) µ µ mol/l), mol/l), DAPT (10 mol/l) and SB-431542 (10 (10 SB-431542 and mol/l) and and Ω Mpz xrsin td i hmn dl tissues. adult human in study expression resulting in in resulting n . DRG explants and DRG neurons were obtained and cultured cultured and obtained were neurons DRG and explants DRG . etics GAPDH µ mol/l) mol/l) and SU5402 (10 52 R , series 5 3 . Total RNA was extracted from tissues and cell cell and tissues from extracted was RNA Total . and entered into line graphs or heat maps using < 10 M 10 < µ mol/l) was added to cells from day 0 to day 4 day to 0 day from cells to added was mol/l) Ω . Whole-cell capacitances were fully fully were capacitances Whole-cell . µ 2 , 1 mM MgCl mol/l) mol/l) were added to promote The human embryonic stem 2 , 2.5 mM MgATP, mM 2.5 mM , 0.5 β -mercaptoethanol) was was -mercaptoethanol) 22 Human adult tissue tissue adult Human , 2 3 . Cells were seeded seeded were Cells . 2 , 10 mM HEPES S100 , , Gfap All All R pip - - - - ,

Fisher, PI32230). Signals were obtained using Luminata Forte Western Western Forte Luminata using obtained were Signals PI32230). Fisher, (Thermo IgG anti-mouse goat HRP-conjugated by followed T9026) Aldrich, anti– mouse and sc-40) (clone Cruz, anti-Myc Santa mouse 9E10, using visualized and Healthcare) (GE membranes Hybond-C onto blotted SDS-PAGE, by separated were Proteins 26. stage at NP-40, 0.1% SDS, 150 deoxycholate, mM 1% NaCl, sodium 25 mM (1% Tris-HCl, pH 7.4) buffer RIPA in prepared were extracts Protein ng/embryo). 20 Myc- embryos. of MO) (Prdm12 20% and MO) (control 5% in lethal was MO injection stage. two-cell at the embryos into Apparatus) Synthesized stage. (Harvard PLI-100 a Pico-Injector with one-cell microinjected mRNAs were MOs and the during 7.8 pH at hydrochloride L-cysteine tively. Embryos were obtained by and fertilization with dejellied artificial 4.6% CAATTTATA-3 control MO and 5 the MO Prdm12 had sequences Tools.Gene from The were obtained (MOs) oligomers antisense Morpholino with the mMESSAGE mMACHINE SP6 kit transcription (Life Technologies). and taining wild-type mutated For the preparation of mRNAs for microinjections, pCS2 plasmid vectors con normal of stages the to correspond study this Xenopus tetrodotoxin. of application the by characterized further were channels ion of Functions ms. 100 for mV 0 to by of currents the stepping from voltage mV −120 a sodium potential holding resolved resolved by SDS-PAGE and blotted onto Protran membranes (GE Healthcare). dation on Prdm12 turnover, we with 5 cells treated and sonicated was resuspended in buffer RIPARIPA buffer. with To explore a extraction potential effect of proteasomalafter degra remaining pellet The transfection. after h 24 lysis RIPAbuffer or in 7.4) pH Tris, mM 10 lysed SDS, (1% were buffer Cells efficiency). transfection for (control plasmid GFP a and constructs expression Prdm12 (Myc-tagged) mouse and (HA-tagged) JetPei Transfection Reagent (Peqlab) was used to co-transfect cells with human containing 10% fetal bovine serum and 2 mM L-glutamine (Life Technologies). mycoplasma but were was Cells in authenticated. cultured not DMEM further Sigma-Aldrich and maintained at low passages. The cell line tested negative for Prdm12. exogenous of levels Expression (Millipore). Westernsubstrate HRP Forte Luminata using obtained were Signals PI32260). Fisher, (Thermo IgG (clone by followed HRP-conjugated goat 07-690) anti-mouse IgG and HRP-conjugated(Millipore, anti-H3K9me2 goat anti-rabbit anti-H3 rabbit mouse and 05-685) sc-40), Millipore, RR202, Cruz, Santa 9E10, (clone mouse with anti-Myc incubation and membranes Hybond-C to transfer using done was by SDS-PAGE. Immunoblotting RIPAresolved were proteins buffer, and in prepared were lysates Cell 15). (stage stage mid-neurula the until cultured and stage blastula the at dissected region was embryos injected ectoderm the of cap) The (animal co-injected. were mRNAs ng/embryo) 1 mutations, cells studies. methylation Histone condition). per embryos live 40 least (at three types main into visual categorized by were patterns determined embryos, was MO-injected for markers inspection; of pattern distribution The substrate. with Harland’s protocol 28) modified a stage with or 26 labeled (stage stage tailbud and late the at performed was polymerase hybridization RNA Whole-mount (Roche). kit T7 labeling RNA DIG the with using digoxigenin synthesized were Probes pBluescript-SK- pCS2p- for riboprobes Whole-mount embryo. per MO Prdm12 or control ng 20 with injected were cranial embryos of markers, placode pattern distribution the of assessment For (Millipore). substrate To confirm the efficiency of with Tothe MO,Prdm12 embryos we the efficiency co-injected confirm 5 Prdm12 6 ; each 0.05 ng/embryo) and and ng/embryo) 0.05 each ; Islet1 embryo manipulation. embryo mRNA (1 ng/embryo) and control or Prdm12 MO (5, 10 and and 10 (5, MO Prdm12 or control and ng/embryo) (1 mRNA pBluescript-SK- , Prdm12 in in situ ′ Six3 and 5 and pCS2- and hybridization of hybridization ′ and cranial placode markers, pBluescript-SK- and placode cranial -GCAGCACCGAGCCCATCATTAA TTC-3 Prdm12

Wnt8 Pax8 Ebf3 The developmental stages mentioned in in mentioned stages developmental The 5 Prdm12 5 pBluescript-SK- , were linearized and used as templates. templates. as used and linearized were using NBT/BCIP as the chromophoric chromophoric the as NBT/BCIP using , , cDNA were and linearized transcribed Chrd Xenopus COS-7 cells were purchased from from purchased were cells COS-7 α (for inducing neural crest–like crest–like neural inducing (for (wild type and CIP-associated CIP-associated and type (wild -tubulin (clone DM1A, Sigma- DM1A, (clone -tubulin Xenopus laevis Xenopus embryos. ′ -CCTCTTACCTCAGTTA µ M MG132. M Proteins MG132. were Ath3 doi: For preparation of 10.1038/ng.3308 development pGEM- , ′ Prdm12 , , respec in situ in Pax6 5 4 - - - , , .

© 2015 Nature America, Inc. All rights reserved. coverslip and cultured in DMEM containing 10% fetal bovine serum and and serum bovine fetal human with HA-PRDM12 for 4 were 24 mM transfected h. L-glutamine Cells 10% containing DMEM in cultured and coverslip 10 × 1.5 of density a at poly–L-lysine with coated onto coverslips glass seeded were Cells authenticated. further not was but mycoplasma for negative tested line cell The passages. low at maintained and Sigma-Aldrich studies. localization Subcellular ECL. using obtained IgG were Signals 7074). and anti-mouse HRP-conjugated goat IgG Signaling, anti-rabbit (Cell rabbit HRP-conjugated by followed 9677) Signaling, (Cell rabbit anti-HA, mouse anti– using detected were Proteins membranes. onto Protran blotted and SDS-PAGE to subjected were Fractions min. 5 for °C 4 at into separated were extracts Cell chromatin-bound and soluble protein 7.5). by fractions atcentrifugation 4,000 rpm pH X-100, Triton 1% DTT, mM 1 HEPES, mM 1 MgCl mM sucrose, (10 mM 300 NaCl, mM 150 buffer CSK in lysed were cells Reagent. transfection, after Transfection day The JetPei using HA-PRDM12 human with transfected fractionation. Chromatin and fraction immunoprecipitation extracts. whole-cell the in G9a in Myc-Prdm12 of amount the to normalized was FLAG-G9a co-immunoprecipitated of amount The ImageJ. with quantified and ECL using obtained were Signals IgG. anti-mouse rabbit HRP-conjugated by followed F3165) Sigma-Aldrich, M2, (clone anti-FLAG mouse and 631206) Clontech, 9E10, (clone anti-Myc mouse using detected resolved by °C, SDS-PAGE 98 at and blotted min onto 10 Hybond-C for membranes. Proteins buffer were sample SDS in eluted were proteins Bound PBS. were Beads overnight. recovered by and centrifugation washed five times with °C 4 at (Clontech) beads agarose A protein to cross-linked anti-Myc mouse after 48 h in lysis buffer (1× PBS, 5 mM EDTA, 0.5% NP-40) and incubated with Prdm12 using JetPei Transfection Reagent. Total cellular protein was collected Myc- mouse and USA) Kentucky, Lexington, Kentucky, of University Dong, above and co-transfected with human FLAG-tagged G9a (a gift from Chenfang G9a. with Prdm12 of Interaction to normalized were Prdm12 and for GFP for signals obtained Signals software. ImageJ using (ECL) chemiluminescence enhanced (GE Healthcare). using Densitometry to quantify Prdm12 expression was performed obtained were Signaling, (Cell Signals IgG 7074). anti-rabbit goat HRP-conjugated and P0260) (Dako, IgG anti-mouse rabbit HRP-conjugated with incubation by followed 2144), anti– rabbit and ab290) (Abcam, anti-GFP rabbit MMS-101P-200), Covance, anti-HA 16B12, mouse (clone 631206), Clontech, Immunoblots were developed by incubation with mouse anti-Myc (clone 9E10, doi: 10.1038/ng.3308 α -tubulin (Cell Signaling, 2144) and rabbit anti-acetylated histone 3 histone anti-acetylated rabbit and 2144) Signaling, (Cell -tubulin α -tubulin. COS-7 cells were cultured as described above and above as described were cultured cells COS-7 HEK-293T cells were purchased from from purchased were cells HEK-293T COS-7 cells were cultured as described described as cultured were cells COS-7 2 , , 1 Mg-ATP,mM 1 EGTA,mM α -tubulin (Cell Signaling, Signaling, (Cell -tubulin 5 cells per per cells

47. 46. 45. 44. 43. at * set Mann-Whitney or a two-sided Welch’s two-sided unpaired, an using calculated was analysis. Statistical 4ij codes accession Bank Data (Protein templates as domain) finger zinc second the (for ZNF406 and domain) PR the (for PRDM9 PRDM1, of tures of model Structural PRDM12. Zeiss). (Carl microscope 510 LSM an using and Technologies) examined (Life reagent antifade in Gold ProLong mounted were cells The A-11060). Technologies, IgG (Life anti-mouse rabbit 546–conjugated Fluor anti-HA and mouse Alexa with 3% and BSA incubated with 4% with paraformaldehyde, 0.2% permeabilized Triton X-100, blocked in 24 were HP h, After fixed DNAthe cells (Roche). reagent X-tremeGene using 56. 55. 54. 53. 52. 51. 50. 49. 48. d

Schwarz, J.M., Cooper,J.M., Schwarz, Seelow,& M. Schuelke, D.N., MutationTaster2:D. mutation affect to predicted polymorphisms human for Accounting S. Henikoff, P.C.& Ng, Y.,Choi, Murphy,G.E., Sims, Miller,A.P.S., Chan, & J.R. functional the Predicting A.K. Nicholas, skin in architecture nodal of Disruption C. Sommer, & C. Werner, K., Doppler, Sato, T., Sasai, N. & Sasai, Y. Neural crest determination by co-activation of co-activation by determination crest Y.Neural Sasai, & N. T.,Sasai, Sato, R.M. Harland, J. Faber, & P.D. Nieuwkoop, E. Arnaud, C. Stendel, Grove, E.A.,Tole, S.,Limon,J.,Yip, L.&Ragsdale,C.W. Thehemoftheembryonic of method general A R. Saiki, & B. Krummel, R., Higuchi, human non-synonymous deleterious predicting SNPdryad: Z. Zhang, & K.C. Wong, I.A. Adzhubei, protein function. protein indels. and substitutions acid amino of effect microcephaly. (2013). 168–176 neuropathies. demyelinating with patients of biopsies and embryos. 1967). USA Sci. Acad. system. nervous peripheral the in Ranvier of node the of integrity the (2010). andis genes Wnt recycling. endosomal to myelination nerve peripheral links ofmultiple expression the by mice. Gli3-deficient in compromised defined is cortex cerebral andDNAinteractions. protein Res. Acids of Nucleic study DNA fragments: of mutagenesis specific (2014). sequences. protein orthologous only using SNPs mutations. age. deep-sequencing the for prediction and and P Zic1 < 0.05, ** 0.05, < 2el genes in genes s Methods Cell Biol. Cell Methods ) using the Swiss-PdbViewer software. Swiss-PdbViewer the using ) et al. et Nat. Methods Nat. t al. et J. Med. Genet. Med. J. In situ In

t al. et t al. et P SH3TC2/KIAA1985 protein is required for proper myelination and myelination proper for required is protein SH3TC2/KIAA1985 106 Genome Res. Genome HT2 a rti mtn i CactMreToh neuropathy, Charcot-Marie-Tooth in mutant protein a SH3TC2, < 0.01 or *** or 0.01 < Data are presented as mean mean as presented are Data

Xenopus 16 , 17528–17533 (2009). 17528–17533 , hybridization: an improved whole-mount method for method whole-mount improved an hybridization: h mlclr adcp of landscape molecular The mto ad evr o peitn dmgn missense damaging predicting for server and method A , 7351–7367 (1988). 7351–7367 ,

7 , 248–249 (2010). 248–249 , ectoderm.

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We PRDM12 modeled using struc the crystal 12 , 685–695 (1991). 685–695 , P -test (nonparametric data). Significance was Significance data). (nonparametric -test , 249–253 (2009). 249–253 , < 0.001. < , 436–446 (2002). 436–446 , Development Development Nat. Methods Nat. PLoS One PLoS Bioinformatics

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