Nanog retrotransposed genes with functionally conserved open reading frames Morag Robertson, Frances Stenhouse, Douglas Colby, Jamie R.K. Marland, Jennifer Nichols, Susan Tweedie, Ian Chambers Centre Development in Stem Cell Biology, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, KingÕs Buildings, West Mains Road, Edinburgh EH9 3JQ, Scotland Received: 10 October 2005 / Accepted: 15 February 2006 Abstract The Nanog gene plays a key role in the pluripotency of early embryonic cells in vitro and in vivo. In this article retrotransposed copies of Nanog, termed NanogPc and NanogPd, are identified on mouse Chromosomes 4 and 7, respectively. In contrast to the two previously characterized mouse Nanog retrogenes that contain multiple frameshifts and point mutations, NanogPc and NanogPd are 98% identical to NANOG within the open reading frame and encode proteins with activity in an embryonic stem cell self-renewal assay. Mutations common to all four retrotransposed genes but distinct from Nanog suggest divergence from a common progeni- tor that appears likely to be Nanog because tran- scripts derived from Nanog but not from the retrogenes are detected in germ-line cells. The pos- sibility that expression of Nanog could be errone- ously attributed to novel cellular sources is suggested by the high homology among Nanog, NanogPc, and NanogPd. Analysis of distinct Mus species suggests that NanogPc and NanogPd arose between divergence of M. caroli and M. spretus and indicates that Nanog retrotransposition events con- tinue to occur at a high frequency, a property likely to extend to other germ-line transcripts. Introduction Embryonic stem (ES) cells are attracting renewed attention because of the promise they offer for the alleviation of disease through cell-based therapies (Smith 1998). To effectively exploit this potential, it will be important to understand fully not only the processes governing ES cell differentiation but also the mechanisms by which a pluripotent ES cell population can be expanded through the process of self-renewal (Chambers and Smith 2004). ES cell pluripotency depends on the activity of extracellular signals, including those that can be supplied by leukemia inhibitory factor (LIF) and bone morphogenetic proteins (BMPs) (Smith et al. 1988; Williams et al. 1988; Ying et al. 2003). In addition, intrinsic mediators of pluripotency include the homeodomain proteins OCT4 (Nichols et al. 1998; Niwa et al. 2000) and NANOG (Chambers et al. 2003; Hatano et al. 2005; Mitsui et al. 2003). Elimi- nation of Oct4 or Nanog from the developing mouse embryo causes peri-implantation defects (Mitsui et al. 2003; Nichols et al. 1998). In the case of Oct4 this is a result of a failure to specify the pluripotent identity of cells that become allocated to the embryo interior (Nichols et al. 1998). Cells allocated to the interior of Nanog )/) embryos do not maintain a pluripotent identity but rather differentiate into primitive endoderm, a derivative of the pluripotent inner cells (Mitsui et al. 2003). In contrast, overex- pression of Nanog in ES cells results in a phenotype in which cells no longer require stimulation with either LIF or BMP to maintain their pluripotency (Chambers et al. 2003; Ying et al. 2003). In the course of characterizing the mouse Nanog gene, we identified two highly related sequences that arose by retrotransposition. In this article we report the sequence and chromosomal location of these retrotransposed genes. We demonstrate that the products of the open reading frames (ORFs) exhibit activity in an ES cell self-renewal assay and explore the possibility that these retrogenes are expressed. The relationship of these retrotransposed sequences to other more degraded retrotransposed Nanog pseudogenes, NanogPa and NanogPb (Booth and Holland 2004), and to Nanog itself is discussed. Nucleotide sequence data reported here are available in the Gen- Bank database under accession numbers DQ358089, DQ358090, DQ358091, DQ358092, DQ358093, DQ358094, DQ358095, DQ358096. Correspondence to: Ian Chambers; E-mail: ichambers@ed.ac.uk 732 DOI: 10.1007/s00335-005-0131-y  Volume 17, 732743 (2006)  Ó Springer Science+Business Media, Inc. 2006