title: Tlx3 promotes glutamatergic neuronal subtype specification through direct interactions with the chromatin modifier CBP.
abstract: Nervous system development relies on the generation of precise numbers of excitatory and inhibitory neurons. The homeodomain transcription factor, T-cell leukemia 3 (Tlx3), functions as the master neuronal fate regulator by instructively promoting the specification of glutamatergic excitatory neurons and suppressing the specification of gamma-aminobutyric acid (GABAergic) neurons. However, how Tlx3 promotes glutamatergic neuronal subtype specification is poorly understood. In this study, we found that Tlx3 directly interacts with the epigenetic co-activator cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-binding protein (CBP) and that the Tlx3 homeodomain is essential for this interaction. The interaction between Tlx3 and CBP was enhanced by the three amino acid loop extension (TALE)-class homeodomain transcription factor, pre-B-cell leukemia transcription factor 3 (Pbx3). Using mouse embryonic stem (ES) cells stably expressing Tlx3, we found that the interaction between Tlx3 and CBP became detectable only after these Tlx3-expressing ES cells were committed to a neural lineage, which coincided with increased Pbx3 expression during neural differentiation from ES cells. Forced expression of mutated Tlx3 lacking the homeodomain in ES cells undergoing neural differentiation resulted in significantly reduced expression of glutamatergic neuronal subtype markers, but had little effect on the expression on pan neural markers. Collectively, our results strongly suggest that functional interplay between Tlx3 and CBP plays a critical role in neuronal subtype specification, providing novel insights into the epigenetic regulatory mechanism that modulates the transcriptional efficacy of a selective set of neuronal subtype-specific genes during differentiation.
authors.affiliation: Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; School of Psychological Science, Health Sciences University of Hokkaido, Sapporo, Hokkaido, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shimomura
authors.affiliation: Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Dharmeshkumar
authors.initials: D
authors.lastname: Patel
authors.affiliation: Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Wilson
authors.affiliation: Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Karl R
authors.initials: KR
authors.lastname: Koehler
authors.affiliation: Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Pharmacology, University of Arizona School of Medicine, Tucson, Arizona, United States of America.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Khanna
authors.affiliation: Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Eri
authors.initials: E
authors.lastname: Hashino
mesh_terms: D000818:Animals; D050882:CREB-Binding Protein / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage / Q000235:genetics; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D044127:Epigenesis, Genetic; D005456:Fluorescent Dyes; D016257:Fura-2; D059330:GABAergic Neurons / Q000166:cytology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D002843:Chromatin; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C490797:Tgif1 protein, mouse; C487377:Tlx3 protein, mouse; C090466:proto-oncogene protein Pbx3; D018698:Glutamic Acid; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse; D016257:Fura-2