Abstract

BackgroundThe homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells (SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp (Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements.ResultsRohu Nanog (LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetically, it was closely related to freshwater counterparts. Protein sequence (386 AA of 42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis- à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements.ConclusionThe molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.

Highlights

  • The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/ proliferation and/or maintenance of spermatogonial stem cells (SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals

  • Identifying overlapping Rapid amplification of cDNA ends (RACE) sequences, we were able to deduce a full-length cDNA of LrNanog is of 1992 nucleotides that comprised an open reading frame (ORF) of 1161 nucleotides along with both 5′-untranslated region (UTR) of 175 nucleotides and 3 ́-UTR of 656 nucleotides

  • The ORF initiated with an ATG start codon fulfilling the consensus Kozak criterion (A/GNNATGG) of Multiple sequence alignments for the deduced fulllength Nanog proteins of available tetrapod and teleosts revealed that LrNanog shared a relatively higher sequence identity with D. rerio and C. auratus

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Summary

Introduction

The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/ proliferation and/or maintenance of spermatogonial stem cells (SSCs) via interacting with transcription factors such as Oct and Sox in mammals. Homeodomain-containing proteins are transcription factors (TFs) that regulate diverse developmental programmess by modulating expression patterns of targeted genes in a temporal, spacial and tissue-specific manner [3]. They are involved in cell identity/proliferation and play a fundamental role in metazoan development [4]. Nanog, belonging to a member of the homeobox family, is believed to be a transcriptional activator It binds to a 5′-TAAT-3′ core DNA motif. It is believed to be associated with-finely tuned mechanistic pathways for maintenance of pluripotency and self-renewal of undifferentiated embryonic stem cells (ESCs) [5], even in the absence of leukaemia inhibitory factor (LIF) [6, 7]. The exact mechanisms by which Nanog is recruited to its binding sites, the mode of distinction between up-regulated and downregulated targets; and the way Nanog signals to the RNA polymerase to either initiate or repress transcription are currently unresolved

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