Abstract
BackgroundNANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs.ResultsWe first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5′ flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG.ConclusionsWe show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.
Highlights
NANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs)
Characterization of the chicken NANOG (cNANOG) core promoter in PGCs and ESCs To investigate the proximal region of the core promoter of the cNANOG gene, we generated a series of 5′ deletion luciferase reporter constructs of the 6− region, which were randomly designed based on the − 3550/+ 70 bp sequence (Fig. 2a)
Luciferase activity derived from differently sized fragments of the cNANOG promoter was examined in PGCs, ESCs, and DF-1 cells transfected with the constructs for 24 h using Lipofectamine 2000
Summary
NANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. We investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs. Gene transcription is mainly regulated by transcription factors (TFs) that bind to specific DNA sequences (called motifs) located in the promoter regions of genes [1]. Identification of regulatory elements within the promoter region is considered crucial to understand the mechanism underlying transcriptional regulation in specific cell types. Germ cells have a unique mechanism of transcription initiation that uses alternate forms of core promoter elements [7,8,9,10]. Germ cells reorganize different type of core promoter TFs under the control of germ cell-specific TFs during germ cell differentiation [11,12,13]
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