ARCHIVAL REPORT Molecular Networks of DYX1C1 Gene Show Connection to Neuronal Migration Genes and Cytoskeletal Proteins Kristiina Tammimies, Morana Vitezic, Hans Matsson, Sylvie Le Guyader, Thomas R. Bu ¨ rglin, Tiina O ¨ hman, Staffan Str ¨ omblad, Carsten O. Daub, Tuula A. Nyman, Juha Kere, and Isabel Tapia-Pa ´ez Background: The dyslexia susceptibility 1 candidate 1 (DYX1C1) gene has recently been associated with dyslexia and reading scores in several population samples. The DYX1C1 has also been shown to affect neuronal migration and modulate estrogen receptor signaling. Methods: We have analyzed the molecular networks of DYX1C1 by gene expression and protein interaction profiling in a human neuroblastoma cell line. Results: We find that DYX1C1 can modulate the expression of nervous system development and neuronal migration genes such as RELN and associate with a number of cytoskeletal proteins. We also show by live cell imaging that DYX1C1 regulates cell migration of the human neuroblastoma cell line dependent on its tetratricopeptide repeat and DYX1 protein domains. The DYX1 domain is a novel highly conserved domain identified in this study by multiple sequence alignment of DYX1C1 proteins recovered from a wide range of eukaryotic species. Conclusions: Our results contribute to the hypothesis that dyslexia has a developmental neurobiological basis by linking DYX1C1 with many genes involved in neuronal migration disorders. Key Words: Dyslexia, interactome, neuronal migration, phylogenetic analysis, protein domain, transcriptome D yslexia, a common impairment in learning to read and write despite normal intelligence and normal senses, affects approximately 5%–10% of the population and has a strong genetic component (1,2). The dyslexia susceptibility 1 candidate 1 (DYX1C1) is one of the promising candidate genes for dyslexia. It was cloned by a study of a familial balanced translocation cosegregating with dyslexia (3), and after the first publication, several independent studies have shown that DYX1C1 is associated with dyslexia, reading/writing ability, or short-term memory (4–10). The function of DYX1C1 is mostly unknown; however, it has been demonstrated to affect neuronal migration and modulate estrogen receptor signaling (11,12). Moreover, RNA interference knockdown of Dyx1c1 in rats leads to impaired auditory processing and spatial learning (13). Recently, DYX1C1 was shown to interact with key proteins in the autophagy system (14). In addition to DYX1C1, a number of other dyslexia candidate genes have been cloned (15), but the neurobiology and molecular networks leading to dyslexia remain mostly undiscovered. Possible mechanisms that might play a role in the development of dyslexia are subtle disturbances in neuronal migration and cortex organiza- tion (16). The hypothesis of abnormal neuronal migration was strengthened after three of the dyslexia susceptibility genes (DYX1C1, DCDC2, and KIAA0319) were suggested to affect neuronal migration (17). Disruption of neuronal migration has also been shown to result in severe developmental disorders such as lissencephaly and epilepsy but also psychiatric disorders such as schizophrenia and bipolar disorder (18). Many genes have been identified to cause neuronal migration disorders—for instance, mutations in Reelin (RELN), Lissen- cephaly 1 (LIS1), and Doublecortin (DCX) (18). In this study, we analyzed molecular networks of DYX1C1 with global transcriptome and protein interaction assays. We demon- strate that DYX1C1 can modulate the expression of genes involved in cell migration and nervous system development and associate with a number of cytoskeletal proteins. In addition, we show that DYX1C1 affects random cell migration of the neuroblastoma SH-SY5Y cells. By these means, we provide thorough knowledge about the cellular networks of DYX1C1 leading to regulation of cell migration and its possible involve- ment in the etiology of dyslexia. Methods and Materials Cell Culture SH-SY5Y neuroblastoma cell line was cultured as described previously (12). The generation of DYX1C1 stable SH-SY5Y cell line (SH-SY5Y DYX1C1 ) is described in detail in the Supplemental Methods and Materials in Supplement 1. Gene Expression Microarrays The microarray experiments were done with Illumina Human HT-12_V4_expression bead arrays in triplicates for all the samples (SH-SY5Y, SH-SY5Y DYX1C1 , siControl, and siDYX1C1) according to the protocol of the supplier and analyzed with R program lumi From the Center for Biosciences (KT, HM, SLG, TRB, SS, COD, JK, IT-P), Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge; Department of Cell and Molecular Biology (MV); Science for Life Laboratory (JK), Karolinska Institutet, Stockholm, Sweden; Omics Science Center (MV, COD), RIKEN Yokohama Institute, Tsurumi-ku, Yokohama, Kanagawa, Japan; Institute of Biotechnology (TO ¨ , TAN), University of Helsinki; and the Folkh¨ alsan Institute of Genetics (JK), Helsinki and Department of Medical Genetics, University of Helsinki, Helsinki, Finland. Address correspondence to Juha Kere, M.D., Ph.D., Department of Biosciences and Nutrition, Karolinska Institutet, H¨ alsov¨ agen 7, Huddinge 14183, Sweden; E-mail: juha.kere@ki.se. Received Feb 11, 2012; revised Aug 7, 2012; accepted Aug 8, 2012. 0006-3223/$36.00 BIOL PSYCHIATRY 2013;73:583–590 http://dx.doi.org/10.1016/j.biopsych.2012.08.012 & 2013 Society of Biological Psychiatry