A Dual Origin of the Xist Gene from a Protein-Coding Gene and a Set of Transposable Elements Eugeny A. Elisaphenko1, Nikolay N. Kolesnikov1, Alexander I. Shevchenko1, Igor B. Rogozin1,2, Tatyana B. Nesterova3, Neil Brockdorff4, Suren M. Zakian1* 1 Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk, Russia, 2 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America, 3 Medical Research Council, Clinical Sciences Centre, Imperial College Faculty of Medicine, London, United Kingdom, 4 Department of Biochemistry, University of Oxford, Oxford, United Kingdom Abstract X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA. Citation: Elisaphenko EA, Kolesnikov NN, Shevchenko AI, Rogozin IB, Nesterova TB, et al. (2008) A Dual Origin of the Xist Gene from a Protein-Coding Gene and a Set of Transposable Elements. PLoS ONE 3(6): e2521. doi:10.1371/journal.pone.0002521 Editor: Sudhindra Gadagkar, University of Dayton, United States of America Received January 22, 2008; Accepted May 15, 2008; Published June 25, 2008 Copyright: 2008 Elisaphenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the Russian Foundation for Fundamental Research (grants no. 06-04-48337-a and 08-04-00346-a), Russian Academy of Sciences foundation program for Molecular and Cellular Biology and by grant 10.8 from the Interdisciplinary Integration Project of the Siberian Division of the Russian Academy of Sciences. I.B.R. was supported in part by the Intramural Research Program of the National Library of Medicine at National Institutes of Health/ DHHS. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zakian@bionet.nsc.ru Introduction The majority of genes on one of the two X chromosomes are inactivated in female mammals during early development. It is postulated that this phenomenon is required to equalize X chromosome gene expression in males (XY) and females (XX) [1]. In eutherians X inactivation is controlled by the X-linked inactivation center (XIC). The XIC is a complex genetic locus comprising of a number of genes producing non-coding nuclear RNAs [2]. Key amongst these is the X inactive specific transcript (Xist), that plays a pivotal role in X inactivation. Xist RNA spreads in cis along the X chromosome to be inactivated, initiates transcriptional silencing, and triggers chromatin modifications that maintain an inactive state of the genes [3]. Duret et al. [4] proposed that the XIC genes Tsx (Testis specific X-linked gene), Xist, Enox(Jpx) (Expressed neighbour of Xist), Ftx (Mus musculus FTX non-coding RNA), and Cnbp2 (Cellular nucleic acid binding protein 2) originated through pseudogeniza- tion of the protein-coding genes Fip1l2 (Polyadenylation factor I complex (Saccharomyces cerevisiae), like 2), Lnx3 (Ligand of numb-protein X 3), Rasl11c (RAS-like, family 11, member C), Uspl (Ubiquitin specific peptidase), and Wave4(Wasf3) (Wiskott-Aldrich syndrome protein family member 3), respectively. This assumption is based on the fact that the genes in question are located within the regions of synteny in chicken (Gallus gallus) and human (Homo sapiens) and contain homologous regions. Xist may have evolved in part from the Lnx3 gene, which in chicken produces an mRNA encoding a protein similar to PDZ domain containing ring finger 1 (LOC422320, ACC XM_420296.1). Lnx3 exons 4 and 11 show similarity to exons h4/m4 and h5/m6 of human and mouse XIST/Xist, respectively. The function of Lnx3 in chicken is unknown, and this gene ceased to exist in eutherians after contributing to Xist formation. Six exons of Fip1l2 show similarity to the orthologous region of human and mouse. Three of them correspond to mouse Tsx or human pseudo-TSX. Four Rasl11c exons are homologous to sequences in the canine and bovine XIC, and one Rasl11c exon displays similarity to a sequence in the human XIC [4]. The contribution of Uspl and Wave4(Wasf3) to the formation of XIC genes remains to be clarified. In this study we have conducted an independent analysis of the origin and evolution of the X inactivation center and the Xist gene in eutherians using a set of bioinformatic approaches. We have compiled a consensus Xist gene reconstructed from sequences that had undergone considerable divergence in each species. As a result, additional data have been obtained demonstrating how the XIC originated from a region containing protein-coding genes. In particular, we have demonstrated that the genes Enox(Jpx) and Ftx of the XIC contain exons homologous to those in cognate protein- coding genes Uspl and Wave4(Wasf3), and we have discovered at least three additional exons of Lnx3 in the Xist gene sequence. Moreover we have found that many Xist exons originated from mobile elements of various classes, which gave rise to simple PLoS ONE | www.plosone.org 1 June 2008 | Volume 3 | Issue 6 | e2521