{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:47:05Z","timestamp":1760150825620,"version":"build-2065373602"},"reference-count":52,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T00:00:00Z","timestamp":1642550400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"SCHWEIZERISCHER NATIONALFONDS ZUR F\u00d6RDERUNG DER WISSENSCHAFTLICHEN FORSCHUNG","award":["31003A_122359"],"award-info":[{"award-number":["31003A_122359"]}]},{"DOI":"10.13039\/100007214","name":"VELUX FOUNDATION","doi-asserted-by":"publisher","award":["1371"],"award-info":[{"award-number":["1371"]}],"id":[{"id":"10.13039\/100007214","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Basic helix\u2013loop\u2013helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and\/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, F\u00f6rster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability.<\/jats:p>","DOI":"10.3390\/ijms23031053","type":"journal-article","created":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T08:20:57Z","timestamp":1642580457000},"page":"1053","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6537-8627","authenticated-orcid":false,"given":"David","family":"Atac","sequence":"first","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9449-2189","authenticated-orcid":false,"given":"Lucas","family":"Mohn","sequence":"additional","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"},{"name":"Neuroscience Center Zurich, University and ETH Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Silke","family":"Feil","sequence":"additional","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1333-7070","authenticated-orcid":false,"given":"Kevin","family":"Maggi","sequence":"additional","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dominik","family":"Haenni","sequence":"additional","affiliation":[{"name":"Center for Microscopy and Image Analysis, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Britta","family":"Seebauer","sequence":"additional","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"},{"name":"Neuroscience Center Zurich, University and ETH Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0965-0539","authenticated-orcid":false,"given":"Samuel","family":"Koller","sequence":"additional","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0370-3815","authenticated-orcid":false,"given":"Wolfgang","family":"Berger","sequence":"additional","affiliation":[{"name":"Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland"},{"name":"Neuroscience Center Zurich, University and ETH Zurich, 8057 Zurich, Switzerland"},{"name":"Zurich Center for Integrative Human Physiology, University of Zurich, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1016\/0092-8674(89)90434-0","article-title":"Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence","volume":"58","author":"Murre","year":"1989","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1038\/nrn874","article-title":"Proneural genes and the specification of neural cell types","volume":"3","author":"Bertrand","year":"2002","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.brainres.2018.03.013","article-title":"bHLH transcription factors in neural development, disease, and reprogramming","volume":"1705","author":"Dennis","year":"2019","journal-title":"Brain Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1021\/bi701527r","article-title":"Crystal Structure of E47\u2212NeuroD1\/Beta2 bHLH Domain\u2212DNA Complex: Heterodimer Selectivity and DNA Recognition","volume":"47","author":"Longo","year":"2008","journal-title":"Biochemistry"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1002\/bies.950131002","article-title":"The helix-loop-helix domain: A common motif for bristles, muscles and sex","volume":"13","author":"Garrell","year":"1991","journal-title":"BioEssays"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1002\/stem.1093","article-title":"Concise Review: Blood Relatives: Formation and regulation of hematopoietic stem cells by the basic helix-loop-helix transcription factors stem cell leukemia and lymphoblastic leukemia-derived sequence 1","volume":"30","author":"Curtis","year":"2012","journal-title":"Stem Cells"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1016\/j.semcdb.2005.07.006","article-title":"MyoD and the transcriptional control of myogenesis","volume":"16","author":"Berkes","year":"2005","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1111\/j.1365-2443.1996.tb00016.x","article-title":"Mash1 promotes neuronal differentiation in the retina","volume":"1","author":"Tomita","year":"1996","journal-title":"Genes Cells"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4821","DOI":"10.1242\/dev.125.23.4821","article-title":"Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis","volume":"125","author":"Brown","year":"1998","journal-title":"Development"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1242\/dev.126.1.23","article-title":"NeuroD regulates multiple functions in the developing neural retina in rodent","volume":"126","author":"Morrow","year":"1999","journal-title":"Development"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"14996","DOI":"10.1073\/pnas.96.26.14996","article-title":"X-ngnr-1 and Xath3 promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina","volume":"96","author":"Perron","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.ydbio.2005.05.041","article-title":"The role of combinational coding by homeodomain and bHLH transcription factors in retinal cell fate specification","volume":"285","author":"Wang","year":"2005","journal-title":"Dev. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"37894","DOI":"10.1074\/jbc.M703616200","article-title":"Highly Conserved Sequences Mediate the Dynamic Interplay of Basic Helix-Loop-Helix Proteins Regulating Retinogenesis","volume":"282","author":"Hernandez","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1007\/s00335-001-2101-3","article-title":"Molecular characterization and mapping of ATOH7, a human atonal homolog with a predicted role in retinal ganglion cell development","volume":"13","author":"Brown","year":"2002","journal-title":"Mamm. Genome"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5675","DOI":"10.1073\/pnas.88.13.5675","article-title":"Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis","volume":"88","author":"Brennan","year":"1991","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4689","DOI":"10.1093\/emboj\/16.15.4689","article-title":"Crystal structure of PHO4 bHLH domain-DNA complex: Flanking base recognition","volume":"16","author":"Shimizu","year":"1997","journal-title":"EMBO J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1016\/S0896-6273(00)80391-8","article-title":"Xath5 Participates in a Network of bHLH Genes in the Developing Xenopus Retina","volume":"19","author":"Kanekar","year":"1997","journal-title":"Neuron"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/S0896-6273(00)00034-9","article-title":"Midline Signals Regulate Retinal Neurogenesis in Zebrafish","volume":"27","author":"Masai","year":"2000","journal-title":"Neuron"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1649","DOI":"10.1073\/pnas.98.4.1649","article-title":"The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development","volume":"98","author":"Liu","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1242\/dev.128.2.217","article-title":"Specification of neurotransmitter receptor identity in developing retina: The chick ATH5 promoter integrates the positive and negative effects of several bHLH proteins","volume":"128","author":"Matter","year":"2001","journal-title":"Development"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1101\/gad.855301","article-title":"Requirement for math5 in the development of retinal ganglion cells","volume":"15","author":"Wang","year":"2001","journal-title":"Genes Dev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2497","DOI":"10.1242\/dev.128.13.2497","article-title":"Math5 is required for retinal ganglion cell and optic nerve formation","volume":"128","author":"Brown","year":"2001","journal-title":"Development"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1016\/S0896-6273(01)00312-9","article-title":"Retinal Ganglion Cell Genesis Requires lakritz, a Zebrafish atonal Homolog","volume":"30","author":"Kay","year":"2001","journal-title":"Neuron"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"578","DOI":"10.1038\/nn.2798","article-title":"Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing NCRNA disease","volume":"14","author":"Ghiasvand","year":"2011","journal-title":"Nat. Neurosci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1093\/hmg\/ddr509","article-title":"Next generation sequencing identifies mutations in Atonal homolog 7 (ATOH7) in families with global eye developmental defects","volume":"21","author":"Khan","year":"2011","journal-title":"Hum. Mol. Genet."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3681","DOI":"10.1093\/hmg\/dds197","article-title":"ATOH7 mutations cause autosomal recessive persistent hyperplasia of the primary vitreous","volume":"21","author":"Prasov","year":"2012","journal-title":"Hum. Mol. Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"462","DOI":"10.3109\/13816810.2015.1120316","article-title":"Mutations in ATOH7 gene in patients with nonsyndromic congenital retinal nonattachment and familial exudative vitreoretinopathy","volume":"37","author":"Kondo","year":"2016","journal-title":"Ophthalmic Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1028","DOI":"10.1167\/iovs.16-20281","article-title":"The Genetic Causes of Nonsyndromic Congenital Retinal Detachment: A Genetic and Phenotypic Study of Pakistani Families","volume":"58","author":"Keser","year":"2017","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_29","first-page":"132","article-title":"Atonal homolog 7 (ATOH7) loss-of-function mutations in predominant bilateral optic nerve hypoplasia","volume":"29","author":"Atac","year":"2019","journal-title":"Hum. Mol. Genet."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2716","DOI":"10.1093\/hmg\/ddq144","article-title":"Genome-wide association identifies ATOH7 as a major gene determining human optic disc size","volume":"19","author":"Macgregor","year":"2010","journal-title":"Hum. Mol. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2464","DOI":"10.1093\/hmg\/ddr120","article-title":"Common genetic variants associated with open-angle glaucoma","volume":"20","author":"Ramdas","year":"2011","journal-title":"Hum. Mol. Genet."},{"key":"ref_32","first-page":"1","article-title":"NLStradamus: A simple Hidden Markov Model for nuclear localization signal prediction","volume":"10","author":"Ba","year":"2009","journal-title":"BMC Bioinform."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1271","DOI":"10.1073\/pnas.89.4.1271","article-title":"Fluorescence lifetime imaging of free and protein-bound NADH","volume":"89","author":"Lakowicz","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/S0962-8924(98)01410-X","article-title":"Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell","volume":"9","author":"Bastiaens","year":"1999","journal-title":"Trends Cell Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"144","DOI":"10.2174\/2211555203666141117221111","article-title":"FLIM-FRET for Cancer Applications","volume":"3","author":"Rajoria","year":"2015","journal-title":"Curr. Mol. Imaging"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1111\/j.1365-313X.2008.03469.x","article-title":"Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis","volume":"55","author":"Groszmann","year":"2008","journal-title":"Plant J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1128\/MCB.16.4.1367","article-title":"Functional domains of the transcription factor USF2: Atypical nuclear localization signals and context-dependent transcriptional activation domains","volume":"16","author":"Luo","year":"1996","journal-title":"Mol. Cell. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1296","DOI":"10.1128\/MCB.00336-06","article-title":"Specific Amino Acid Residues in the Basic Helix-Loop-Helix Domain of SRC-3 Are Essential for Its Nuclear Localization and Proteasome-Dependent Turnover","volume":"27","author":"Li","year":"2007","journal-title":"Mol. Cell. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.bbrc.2006.06.163","article-title":"Nuclear localization of coactivator RAC3 is mediated by a bipartite NLS and importin \u03b13","volume":"348","author":"Yeung","year":"2006","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1016\/j.yexcr.2009.02.025","article-title":"Synergistic nuclear import of NeuroD1 and its partner transcription factor, E47, via heterodimerization","volume":"315","author":"Mehmood","year":"2009","journal-title":"Exp. Cell Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1038\/365661a0","article-title":"The GTP-binding protein Ran\/TC4 is required for protein import into the nucleus","volume":"365","author":"Moore","year":"1993","journal-title":"Nature"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1649","DOI":"10.1083\/jcb.123.6.1649","article-title":"Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran\/TC4 as an essential transport factor","volume":"123","author":"Melchior","year":"1993","journal-title":"J. Cell Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1128\/MCB.20.2.429-440.2000","article-title":"Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms","volume":"20","author":"Massari","year":"2000","journal-title":"Mol. Cell. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1016\/0092-8674(90)90088-V","article-title":"The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation","volume":"60","author":"Davis","year":"1990","journal-title":"Cell"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/j.devcel.2015.10.019","article-title":"E Proteins and ID Proteins: Helix-Loop-Helix Partners in Development and Disease","volume":"35","author":"Wang","year":"2015","journal-title":"Dev. Cell"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"10029","DOI":"10.1128\/MCB.25.22.10029-10039.2005","article-title":"The Basic Domain of ATH5 Mediates Neuron-Specific Promoter Activity during Retina Development","volume":"25","author":"Ballivet","year":"2005","journal-title":"Mol. Cell. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1016\/S0076-6879(96)66024-8","article-title":"Using CLUSTAL for multiple sequence alignments","volume":"Volume 266","author":"Higgins","year":"1996","journal-title":"Methods in Enzymology"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2558","DOI":"10.1093\/bioinformatics\/btm377","article-title":"Comparative protein structure modeling by combining multiple templates and optimizing sequence-to-structure alignments","volume":"23","author":"Rai","year":"2007","journal-title":"Bioinformatics"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"W363","DOI":"10.1093\/nar\/gkm341","article-title":"M4T: A comparative protein structure modeling server","volume":"35","author":"Rai","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/nmeth.2413","article-title":"A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum","volume":"10","author":"Shaner","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"2548","DOI":"10.1021\/acs.jpclett.7b00960","article-title":"Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation","volume":"8","author":"Molina","year":"2017","journal-title":"J. Phys. Chem. Lett."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"20889","DOI":"10.1038\/srep20889","article-title":"Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting","volume":"6","author":"Bajar","year":"2016","journal-title":"Sci. Rep."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/3\/1053\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:03:49Z","timestamp":1760133829000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/3\/1053"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,19]]},"references-count":52,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["ijms23031053"],"URL":"https:\/\/doi.org\/10.3390\/ijms23031053","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2022,1,19]]}}}