Aromatic Residues in the C-terminal Domain 2 Are Required for Nanog to Mediate LIF-independent Self-renewal of Mouse Embryonic Stem Cells

Abstract

Nanog was identified by its ability to sustain the LIF-independent self-renewal of mouse embryonic stem (ES) cells and has recently been shown to play a role in reprogramming adult fibroblasts into pluripotent stem cells. However, little is known about the structural basis of these remarkable activities of Nanog. We have previously identified an unusually strong transactivator named CD2 at its C terminus. Here we demonstrate that CD2 is required for Nanog to mediate ES cell self-renewal. Furthermore, deletion and point mutation analysis revealed that CD2 relies on at least seven aromatic amino acid residues to generate its potent transactivating activity. A mutant Nanog bearing alanine substitutions for these seven residues fails to confer LIF-independent self-renewal in mouse ES cells. Substitution of CD2 by the viral transactivator VP16 gave rise to Nanog-VP16, which is 10 times more active than wild-type Nanog in ES cells. Surprisingly, the expression of Nanog-VP16 in mouse ES cells induces differentiation and is thus unable to sustain LIF-independent self-renewal for mouse ES cells. Taken together, our results demonstrate that the CD2 domain of Nanog is a unique transactivator that utilizes aromatic residues to confer specific activity absolutely required for ES self-renewal.

The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates Stem Cell Proliferation but Not Pluripotency

“…Reporter assays demonstrated that deletion of WR had little effect on transcription activity of Nanog, whereas removal of CD2 reduced its activity severely, suggesting that WR is dispensable for Nanog function (15,16). Consistently, we demonstrated that CD2, apparently endowed by a few critical aromatic residues with its strong transactivation activity, is required for Nanog to mediate LIF-independent ES cell self-renewal (17). Unexpectedly, we recently proved that WR plays an important role in Nanogmediated LIF-independent ES cell self-renewal despite its apparent lack of contribution to Nanog transcription activity.…”

“…Functionally, we were able to assign WR and CD2 as transactivators embedded in its C terminus (15,16). Our further analysis has demonstrated that CD2 is the dominant transactivator and that its activity is absolutely required for Nanog-mediated LIF-independent ES cell self-renewal (17). We report here that WNAAP from the WR domain appears to bind to Nac1 and impact the cell cycle machinery rather than the pluripotency maintenance or self-renewal function of ES cells.…”

Alternative Splicing Produces Nanog Protein Variants with Different Capacities for Self-renewal and Pluripotency in Embryonic Stem Cells

“…The protein domain structure of Nanog has been studied extensively to elucidate its molecular mechanism of action. The C-terminal domain, including the tryptophan-rich (WR) domain, has been deemed essential for the function of Nanog (25)(26)(27)(28)(29); however, study of the N-terminal domain (NTD) has been largely overlooked to date.…”

The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

“…2 g of total RNA was reverse transcribed in a final volume of 20 L as previously described [7] . Real-time reverse-transcriptase (RT)-PCR reactions were performed using the Real-time PCR Master Mix (SYBR GREEN) Reagent Kit (TOYOBO, QPK-201), according to the manufacturer's protocol.…”

“…Both the WR and CD2 are essential for the transcriptional activity and the maintenance of ES cell self-renewal in vivo [6][7][8] . However, the function of the ND has not been reported yet.…”