Context-Dependent Functions of NANOG Phosphorylation in Pluripotency and Reprogramming

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.

Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self‐renewal

“…S130 and S131 correspond to residues 35 and 36 in the canonical homeodomain numbering system [44]. Therefore, the phosphorylation sites identified here by mass spectrometry include residues previously reported as phosphorylation sites [19,45,46] as well as novel sites of phosphorylation within the homeodomain; these are highlighted on a domain diagram of NANOG (Fig. 1B).…”

“…Of the remaining sites, residues 68 and 71 have serines at the −3 position which could act as priming sites in vivo. Consistent with this possibility, serine 65 has been reported to be phosphorylated in cells [19,45]. The probability of serine 71 being phosphorylated by CKI is low (Table 1), and furthermore, there has been no in vivo identification of a potential priming phosphorylation on serine 68.…”

“…We also identified additional CKI sites that have been reported as sites of phosphorylation. Specifically, phosphorylation of mouse NANOG at S56/S57 has been demonstrated in ESCs and in 293T cells [19,45]. In addition, protein kinase A can phosphorylate recombinant human NANOG on the residue equivalent to S78 in mouse NANOG [46].…”

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency

“…These compounds are commonly used to facilitate reprogramming and pluripotent cell culture ( Dakhore et al., 2018 ), and iPSCs generated via 3c enhanced reprogramming are developmentally highly competent ( Amlani et al., 2018 ). 3c enhanced reprogramming yields approximately 50 times more stable iPSC colonies than basal reprogramming and does so in a shorter period of time (6 rather than 12 days of OKSM expression) ( Penalosa-Ruiz et al., 2019 ; Saunders et al., 2017 ; Schwarz et al., 2018 ; Stelzer et al., 2015 ; Vidal et al., 2014 ). Since H3K9 methylation is a well-known roadblock for cellular reprogramming, we speculated that 3c might enhance iPSC formation by counteracting H3K9 methylation more efficiently than the OKSM factors alone.…”

Context-dependent requirement of H3K9 methyltransferase activity during cellular reprogramming to iPSCs

“…These compounds are commonly used to facilitate reprogramming and pluripotent cell culture (Dakhore et al, 2018) and iPSCs generated in this manner are developmentally fully competent (Amlani et al, 2018). 3c reprogramming yields approximately 50 times more stable iPSC colonies than basal reprogramming and does so in a shorter period of time (six rather than twelve days of OKSM expression) (Penalosa-Ruiz et al, 2019;Saunders et al, 2017;Schwarz et al, 2018;Stelzer et al, 2015;Vidal et al, 2014). We therefore speculated that 3c might enhance iPSC formation by counteracting H3K9 methylation more efficiently than OKSM factors alone.…”