Characterization Of Porcine Nanog Gene And Its Up-and Down-stream Regulation Networks

The derivation of porcine pluripotent stem cells has vast importance to animal reproduction and application for the translational medicine.Because bona fide pig embryonic stem cells(ESCs)were not available yet from pig embryos,an alternative to ESCs is the induced pluripotent stem cells generated by reprogramming somatic cells with ectopic expression of pluripotent transcription factors Oct4/Sox2/Klf4/c-Myc.Porcine induced pluripotent stem cells(piPSCs)have been reported by many laboratories worldwide including our laboratory.The presented study focused on the regulation mechanism and gene structure of pluripotency gene NANOG,and the upstream signaling pathway Activin-SMAD as well as the target ESRRB in the porcine pluripotent cell maintenance.The results showed below: 1.Structure and functional evaluation of porcine NANOGNanog contains Homeobox domain and plays an important role in maintaining the pluripotency of murine and human embryonic stem cells.Here,we report,for the first time,that porcine NANOG is encoded by a single exon gene(SEG)mapped on chromosome 1 and has two daughter genes,one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1.The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG,but does not encode a functional protein due to deletions and in-frame stop codons.The NANOGP1 contains four exons and three introns,but is short of the homeodomain sequence.Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG,but not from NANOGP1,because the NANOGP1 promoter is highly methylated,as confirmed by global DNA methylation analysis.The NANOG protein encoded by NANOG retains N,H,and C1/W/C2 domains.The H domain is required for nuclear translocation,while the C1/W/C2 domain ensures the NANOG regulatory function.Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2,KLF4,and c-MYC.In conclusion,the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2,and can promote the activation of target pluripotent factors in vivo.2.ESRRB plays a crucial role in the promotion of porcine cell reprogrammingThe estrogen-related receptor b(ESRRB)is an orphan nuclear receptor and targets many genes involved in self-renewal and pluripotency.In this study,we revealed the expression profile of porcine ESRRB in both pluripotent cells and early stage embryos and dissected the functional domains of ESRRB protein to prove that ESRRB is a key transcription factor and is required for the inner cell mass of the blastocyst and porcine iPS cells.Addition of ESRRB into the cocktail of core pluripotent factors Oct4,Sox2,Klf4,and c-Myc(OSKM+E)could significantly enhance the reprogramming efficiency and the formation of alkaline phosphatase positive colonies.Conversely,knockdown of ESRRB in piPSCs significantly reduced the expression level of pluripotent genes,minimized the alkaline phosphatase activity,and initiated the differentiation of piPSCs.Therefore,porcine ESRRB is a crucial transcription factor for improving the pluripotency of porcine iPS cells.3.Characterization and Functional Analysis of Porcine Estrogen-Related Receptors and Their Alternative Splicing VariantsEstrogen-related receptors(ESRRs)are orphan nuclear hormone receptors with unidentified ligands;they play important roles in tissue regulation and development as well as maintenance of pluripotent cell identity.The splicer variant,genomic organization and physiological roles of ESRRs have been elucidated in human and mouse.However,in livestock they remain elusive.In this study,we cloned porcine ESRR family members ESRRA,ESRRB,and ESRRG.Two ESRRA alternative splicing variants ESRRA1 and ESRRA2 and a novel ESRRG were identified.To determine the domain function,we constructed vectors with sequential deletions of the ESRRB coding sequence.The functional analysis showed that the C domain of ESRRs plays a core role in promoting the activation of estrogen response elements that are found in all kinds of ESRR-targeting genes,while the E domain is not essential for transcription regulation of ESRRs.4.Activin-SMAD signaling is required for maintenance of porcine iPS cell self-renewal through upregulation of NANOG and OCT4 expressionPorcine induced pluripotent stem cells(piPSCs)retain the enormous potential for farm animal reproduction and translational medicine,and have been reported by many laboratories worldwide.Some piPSC lines were bFGF-dependence and showed mouse EpiSC-like morphology;other lines were LIF-dependence and showed mouse ESC-like morphology.Metastable state of piPSC line that required both LIF and bFGF was also reported.Because bona fide pig embryonic stem cells(pESCs)was not available,uncovering piPSC state-specific regulatory circuitries was the most important task.In this study,we explored the function of Activin-SMAD signaling pathway and its downstream activated target genes in piPSCs.Transcriptome analysis showed that genes involved in Activin-SMAD signaling pathway,but not BMP signaling pathway,were activated during porcine somatic cell reprogramming,no matter piPSCs were LIF-or bFGF-dependent.Addition of Activin A or overexpression of SMAD2/3 significantly promoted expressions of porcine NANOG and OCT4,whereas inhibition of Activin-SMAD signaling by SB431542 or SMAD7 reduced NANOG and OCT4 expressions,and induced piPSC differentiation exiting from pluripotent state.Our data demonstrate that activation of Activin-SMAD signaling pathway by addition of Activin A,but not BMP4,in culture medium is necessary for maintenance of self-renewal in porcine pluripotent stem cells.