Abstract
Nanog is a newly identified divergent homeodomain protein that directs the infinite propagation and sustains the pluripotency of embryonic stem cells. It has been reported that murine Nanog has two potent transactivation domains in N-terminal and C-terminal regions. Human Nanog (hNanog) polypeptide shares about 58% and 87% identity to the open reading frame and homeodomain of murine Nanog, respectively. However, the functional domains and molecular mechanisms of hNanog are poorly understood. In this study, for the first time, we presented that only C-terminus of hNanog contains a potent transactivation domain. Based on the amino acid sequences of homeobox domain, we roughly divided hNanog open reading frame into the three regions such as N-terminal, homeodomain and C-terminal regions and constructed either the fusion proteins between hNanog individual and Gal4 DNA binding domain or the context of native hNanog protein. Reporter assays by using reporter plamid containing Gal4 or Nanog binding site revealed that the only C-terminal region exhibited the significant fold induction of transactivation. However, interestingly, there was no significant activation through N-terminal region unlike murine Nanog, suggesting that C-terminal region may have more critical roles in the transcriptional activation of target genes. Taken together, the finding of a putative transactivation domain in hNanog may contribute to the further understanding of molecular mechanism on the regulation of downstream genes involved in self-renewal and pluripotency of human stem cells.
Highlights
The capability for self-renewal and the pluripotency of embryonic stem (ES) cells seem to be under the control of multiple transcriptional factors (Pesce and Scholer, 2001; Pan et al, 2002) in the presence of leukemia inhibitory factor (LIF)
C-terminal region to the homeodomain contains novel Trp (W) repeat motif of unknown structure and function. They reported that both N- and C-terminal regions to the homeodomain in the murine Nanog have active transcriptional activities and that the C-terminal region unexpectedly seems to be more active than the N-terminal region (Pan and Pei, 2003). Hum an Nanog (hNanog) shares about 57% overall and 87% identity within the homeodomain to the murine Nanog
For the first time, we report that only Cterminal region of hNanog possesses the potent transcriptional activity
Summary
The capability for self-renewal and the pluripotency of embryonic stem (ES) cells seem to be under the control of multiple transcriptional factors (Pesce and Scholer, 2001; Pan et al, 2002) in the presence of leukemia inhibitory factor (LIF). Nanog, a member of the homeobox family of DNA binding transcription factors, was identified as a possible candidate for the LIF/Stat3-independent factor for the pluripotency of ES cells. It does block endodermal differentiation and maintain the pluripotency of ES cells even in the absence of LIF (Chambers et al, 2003; Mitsui et al, 2003). C-terminal region to the homeodomain contains novel Trp (W) repeat motif of unknown structure and function They reported that both N- and C-terminal regions to the homeodomain in the murine Nanog have active transcriptional activities and that the C-terminal region unexpectedly seems to be more active than the N-terminal region (Pan and Pei, 2003). Our study indicates that N-terminal region of hNanog does not exhibit the significant transcriptional activity, unlike murine Nanog
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