Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

Abstract

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering. Youth) is a key transcription factor regulating pluripotency in mammalian early embryos and pluripotent stem cells. Cooperating with other master regulators of pluripotency, NANOG plays a central role in pluripotency (1-3) and forms autoregulatory loops to maintain ES cell (ESC) identity (4-7). NANOG was initially identified from its ability to confer mouse (m)ESC selfrenewal without dependence on leukemia inhibitory factor (LIF) when overexpressed in mESCs (8, 9). Disruption of the NANOG gene in mESCs compromises their pluripotency (8); however, mESCs can maintain their self-renewal without NANOG (10). NANOG expression marks fully reprogrammed induced pluripotent stem cells (iPSCs) during reprogramming mammalian somatic cells into a pluripotent state (11); however, NANOG is dispensable for generating iPSCs from somatic cells both exogenously (12) and endogenously (13,14). In contrast, NANOG robustly promotes reprogramming of epiblast stem cells (EpiSCs) (15,16) or human iPSCs (HiPSCs), which have primed state pluripotency with distinct gene expression patterns and cell signaling dependence, into ground-state pluripotency (17)(18)(19)(20).NANOG is composed of 305 amino acids, including a central homeodomain (HD). HDs are ∼60 amino acid DNA-bin...

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

“…We selected NANOG as our first target gene because its deficiency is sufficient to give an immediate readout, as indicated by a clear loss of pluripotent cell morphology (Hayashi et al, 2015). In general, Cas9 can disrupt gene function at any given exon (Doench et al, 2014) while dCas9-KRAB knocks down gene expression only when gRNAs are targeted to the transcription start site (TSS) (Gilbert et al, 2014).…”

Context-Dependent Functions of NANOG Phosphorylation in Pluripotency and Reprogramming

“…Therefore, loss of phosphorylation may have endowed NANOG S65A with an altered structure more conducive to association with those nuclear pluripotency regulators, leading to functional activation of the pluripotency program in reprogramming. Future studies applying X-ray crystallography to solve the full-length WT and S65A NANOG protein structures are warranted to confirm this hypothesis, which is currently a challenge in the field (Hayashi et al., 2015, Jauch et al., 2008). Alternatively, the preferential associations of S65A NANOG with these pluripotency regulators (Figure 4G) may also be due to their subtle increased protein levels that can only be detected by quantitative SILAC IP-MS we have employed.…”

Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program

“…mEpiSCs are converted to mESClike cells only after several passages on mouse embryo fibroblast (MEF) feeders in LIF-containing medium (6). This LIF-dependent conversion can be enhanced by overexpression of transcription factors NANOG, Kruppel-like factor (KLF) 2, KLF4, and/or PR domain 14 (PRDM14), but the conversion efficiencies remain low (4,(7)(8)(9). The difficulty in converting primed cells to naive cells may reflect epigenetic barriers that inhibit conversion (10) or may indicate that the relevant signaling molecules and their interactions remain to be elucidated.…”