Chicken NANOG self‐associatesviaa novel folding‐upon‐binding mechanism

Abstract

NANOG plays a pivotal role in pluripotency acquisition and lineage specification in higher vertebrates, and its expression is restricted to primordial germ cells (PGCs) during early embryonic development. Mammalian NANOG self-associates via conserved tryptophan-repeat motifs in the C-terminal domain (CTD) to maintain pluripotency. Avian NANOG, however, lacks the conserved motifs, and the molecular mechanism underlying the biologic function is not clearly understood. Here, using spectroscopic and biochemical methods as well as cell-based assays, we report that chicken NANOG (cNANOG) oligomerizes through its CTD via a novel folding-upon-binding mechanism. The CTD of cNANOG is disordered as a monomer and associates into an α-helical multimer driven by intermolecular hydrophobic interactions. Mutation of key aromatic residues in the CTD abrogates the self-association, leading to a loss of the proliferation of chicken PGCs and blastoderm cells. Our results demonstrate that the CTD of cNANOG belongs to a novel IDP that switches into a helical oligomer via self-association, enabling the maintenance of PGCs and blastoderm cells.-Choi, H. J., Kim, I., Lee, H. J., Park, Y. H., Suh, J.-Y., Han, J. Y. Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

A novel F‐box domain containing cyclin F like gene is required for maintaining the genome stability and survival of chicken primordial germ cells

“…53,[74][75][76] In addition, the rate of spontaneous mutation on the HPRT locus was detected at a high level in cancer cells as well as in a cell line carrying mutations on the DNA repair pathway genes. 40,61,80 Moreover, the expression of NANOG itself regulated epigenetically in chicken PGCs for maintaining the integrity of cells. The levels of DNA damage response genes, in particular, ATR and H2AFX were increased since 2 hours after knockdown of cyclin F like in chicken PGCs.…”

“…Although, exogenous shRNAs are notoriously unstable and easily degraded by serum proteins, they act as endogenous interfering RNAs and lead to more efficient downregulation of target gene when compared to exogenous siRNAs . NANOG, is one of the core pluripotency related transcription factors, required for the activation of transcription from many genes during the early embryonic development in mammals and many vertebrates . Moreover, the expression of NANOG itself regulated epigenetically in chicken PGCs for maintaining the integrity of cells .…”

“…The constructed vectors were delivered into PGCs using Lipofectamine 2000. For knockdown, NANOG ‐specific siRNA (sense: 5′‐AAC UGU GUG AUA CUA UCA GUU‐3′) was designed against unique regions and transfected into PGCs using Lipofectamine RNAiMAX . After initiated the overexpression or knockdown of NANOG in PGCs, part of cells was collected at every 6 hours intervals for up to 24 hours, and subjected for the total RNA and cDNA preparation.…”

Differential Transcriptional Regulation of the NANOG Gene in Chicken Primordial Germ Cells and Embryonic Stem Cells

“…PGCs express several pluripotency-related TFs such as NANOG, POU5F3, and SOX2, and their expression controls transcription of germness-related genes in these cells (6,22). During early germ cell development, NANOG plays an essential role as a key TF required for formation of PGCs and maintenance of early germ cells (23,24). NANOG-de cient PGCs reportedly undergo apoptotic death (25).…”

“…NANOG is expressed during chicken intrauterine embryonic development and is exclusively expressed in PGCs from Hamburger and Hamilton stage 5 (HH5) to HH8. Therefore, NANOG is also important to maintain pluripotency and cell proliferation in chicken intrauterine embryos and PGCs (24,28,31).…”