The evolving doublecortin (DCX) superfamily

BMC Genomics The evolving doublecortin (DCX) superfamily Orly Reiner 2 Frdric M Coquelle 2 3 Bastian Peter 1 Talia Levy 2 Anna Kaplan 2 Tamar Sapir 2 Irit Orr 0 Naama Barkai 2 Gregor Eichele 4 Sven Bergmann 1 2 0 Department of Biological Services, Weizmann Institute of Science , Rehovot , Israel 1 Department of Medical Genetics, University of Lausanne , Switzerland 2 Department of Molecular Genetics, Weizmann Institute of Science , Rehovot , Israel 3 CNRS - UMR 6026, Universite de Rennes 1, Equipe SDM, Campus de Beaulieu - Bat. 13, 35042 Rennes cedex , France 4 Max-Planck Institute , Hannover , Germany Background: Doublecortin (DCX) domains serve as protein-interaction platforms. Mutations in members of this protein superfamily are linked to several genetic diseases. Mutations in the human DCX gene result in abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, and DCDC2 has been associated with dyslectic reading disabilities. Results: The DCX-repeat gene family is composed of eleven paralogs in human and in mouse. Its evolution was followed across vertebrates, invertebrates, and was traced to unicellular organisms, thus enabling following evolutionary additions and losses of genes or domains. The N-terminal and C-terminal DCX domains have undergone sub-specialization and divergence. Developmental in situ hybridization data for nine genes was generated. In addition, a novel co-expression analysis for most human and mouse DCX superfamily-genes was performed using high-throughput expression data extracted from Unigene. We performed an in-depth study of a complete gene superfamily using several complimentary methods. Conclusion: This study reveals the existence and conservation of multiple members of the DCX superfamily in different species. Sequence analysis combined with expression analysis is likely to be a useful tool to predict correlations between human disease and mouse models. The subspecialization of some members due to restricted expression patterns and sequence divergence may explain the successful addition of genes to this family throughout evolution. - Background Mutations in the X-linked gene doublecortin (DCX) result in lissencephaly or subcortical band heterotopia (SBH) [1,2] (see review in [3]). Lissencephaly is a severe brain malformation characterized by absent (agyria) or decreased (pachygyria) convolutions accompanied by thickening of the cortex [4]. SBH is a related disorder in which there are bilateral bands of gray matter interposed in the white matter between the cortex and the lateral ventricles [5]. In general, lissencephaly and SBH are neuronal migration disorders. SBH is very common among females with mutations in DCX [1,2]. DCX [6-9] and its closely related gene product DCLK (doublecortin-like kinase) [10,11] are classical microtubule (MT) associated proteins (MAPs), and contain two evolutionary conserved tubulin binding repeat sequences. We have initially defined the evolutionary conservation based on a limited number of proteins [12], following the expansion in available sequences, but the recent explosion of available sequence data, strongly warrants revisiting of this issue of the evolution of the DCX protein family. Interestingly, most missense mutations that are found in lissencephaly patients cluster in the defined DCX (doublecortin) domain [12,13]. These tandem repeat protein motifs are 11 kDa and are located in the N-terminal part of the 30 kDa DCX protein. The structure of the DCX domain markedly differs from that of classical MAPs adopting a globular structure with a ubiquitin-like fold [14]. Cryo-electron microscopy studies revealed that DCX binds to MTs at a novel site, located in between the protofilaments [15]. This binding site can explain why MTs are stabilized by DCX. Two additional genes of this superfamily are implicated in human diseases. RP1 is the protein product of retinitis pigmentosa -1 (RP1) [16,17]. Mutations in this gene result in progressive blindness, not only in humans, but also in a mouse model [18]. DCDC2 has been implicated in dyslexia [19,20]. Reduction of Dcdc2 in the developing cortex using in utero electroporation inhibited neuronal migration [19]. The notion that DCX domain proteins may function in a redundant way during cortical development was raised when Dcx null mice exhibited a very mild cortical phenotype [21], whereas acute knockdown of Dcx using RNAi resulted in a doublecortex phenotype [22]. Recent experimental evidence indicated that DCLK was redundant with DCX. Dclk -/- mice also exhibited a rather mild cortical phenotype, but Dcx/Dclk double mutant mice displayed severe cortical defects [23,24]. Recently, an additional member of this protein family, doublecortin kinase 2 (or DCLK2), has been described, and found to posses MT binding activities [25]. Nevertheless, Dcx and Dclk are but two genes among eleven DCX domain proteins in the mouse genome [26]. Thus, the quest for how DCX domain-containing proteins exert their multiple functions during cortical development is still open [27]. Our previous study has demonstrated common and unique activities in regard to protein interactions, MAP activity, and the subcellular localization of nine of the mouse DCX domain superfamily members [26]. Two unifying functional aspects were detected; all proteins affected MT polymerization, and all interacted with the JNK scaffold proteins, JIP1 and JIP2. Thus, the DCX superfamily of proteins is likely to mediate signal transduction pathways. Proteins with a tandem DCX domain stabilized MTs in transfected cells. Some of the transfected proteins exhibited localization to actin-rich subcellular structures, and in addition most of the DCX domain proteins exhibited a nuclear localization. A distinct set of proteins interacted with the scaffold protein neurabin 2, which binds to PP1 [28] as well as to actin [29]. Positive interactions were observed with DCX, DCLK, and DCLK2, closely related family members, but not with other DCX-domain containing gene products. In the current study, we studied the evolution of the DCX superfamily from unicellular organisms to humans. It was possible to demonstrate emergence and disappearance of specific genes or individual domains. The N-terminal and C-terminal DCX domains have undergone sub-specialization and divergence. In addition, the expression pattern of gene members was studied using database mining and in situ hybridization tools. The sub-specialization of some members due to restricted expression patterns and sequence divergence may explain the successful addition of genes to this family throughout evolution. Results Identification of proteins with a DCX domain Human and mouse proteomes were searched for sequences similar to that of the human DCX domain yielding a total of 22 proteins containing one or two DCX repeats (Table 1, the complete sequences used in the present study are (...truncated)