Abstract
SummaryThe core pluripotency transcription factor NANOG is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Although NANOG is phosphorylated at multiple residues, the role of NANOG phosphorylation in ESC self-renewal is incompletely understood, and no information exists regarding its functions during reprogramming. Here we report our findings that NANOG phosphorylation is beneficial, although nonessential, for ESC self-renewal, and that loss of phosphorylation enhances NANOG activity in reprogramming. Mutation of serine 65 in NANOG to alanine (S65A) alone has the most significant impact on increasing NANOG reprogramming capacity. Mechanistically, we find that pluripotency regulators (ESRRB, OCT4, SALL4, DAX1, and TET1) are transcriptionally primed and preferentially associated with NANOG S65A at the protein level due to presumed structural alterations in the N-terminal domain of NANOG. These results demonstrate that a single phosphorylation site serves as a critical interface for controlling context-dependent NANOG functions in pluripotency and reprogramming.
Highlights
The phospho-proteome in pluripotent stem cells has been extensively and systematically studied (Van Hoof et al, 2012), and has uncovered phosphorylated residues on pluripotency factors that play important roles in establishing and maintaining pluripotency
Studies coupling immunoprecipitation with mass spectrometry (IP-MS) have found that human NANOG is phosphorylated at 11 different sites by ERK2 and CDK1 in human embryonic stem cells (ESCs) (Brumbaugh et al, 2014), and that mouse NANOG is phosphorylated at four different sites (Li et al, 2011; Moretto-Zita et al, 2010) by ERK1 as well as unidentified kinases (Kim et al, 2014)
NANOG Is Phosphorylated at Ser56/57 and Ser65 in mouse ESCs (mESCs) We performed IP-MS of endogenous NANOG in J1 mESCs (Figure 1A), and identified S56/57 and S65 as phosphorylated residues in the N terminus of NANOG (Figure 1B)
Summary
The phospho-proteome in pluripotent stem cells has been extensively and systematically studied (Van Hoof et al, 2012), and has uncovered phosphorylated residues on pluripotency factors that play important roles in establishing and maintaining pluripotency. One study suggested that phosphorylation is important for maintaining NANOG stability in ESCs (Moretto-Zita et al, 2010). This study relied on ectopic expression of NANOG in HEK293 cells for identification of phosphorylation sites by IP-MS, and tested the functions of these phosphorylation sites with phospho-dead or phospho-mimic mutants in the presence of endogenous NANOG in wildtype (WT) mouse ESCs (mESCs) (Moretto-Zita et al, 2010). Our findings contribute important functional data to the phospho-proteome in pluripotent stem cells, and improve our understanding of the key pluripotency regulator NANOG in controlling ESC pluripotency and somatic cell reprogramming
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