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Requirement of Nanog Dimerization for Stem Cell Self-Renewal and Pluripotency

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Requirement of Nanog Dimerization for Stem Cell Self-Renewal and Pluripotency Correction DEVELOPMENTAL BIOLOGY. For the article ‘‘Requirement of Nanog vertical lines, multiple blots from the same experiments were dimerization for stem cell self-renewal and pluripotency,’’ by juxtaposed in Fig. 1A, and irrelevant lanes were removed from Jianlong Wang, Dana N. Levasseur, and Stuart H. Orkin, which the same blot in Figs. 1B,3D, and 4B. In accordance with PNAS appeared in issue 17, April 29, 2008, of Proc Natl Acad Sci USA editorial policy on the preparation of digital images, the cor- (105:6326–6331; first published April 24, 2008; 10.1073͞ rected figures and legends appear below. These changes do not pnas.0802288105), the authors note that, as indicated by thin affect the conclusions of the article. Void ND HD CD A 4MD 700kD 440kD 232kD 158kD 63kD A N Tethered ND HD CD Nanog-Nanog N (NN) GT(GGGS)4GGGT Anti-Nanog ND HD CD N Tethered 100kD 63kD 30kD Nanog-NH NH ND HD Anti-Nanog (NNH) GT(GGGS)4GGGT dimer monomer B NanogV5 FL-Nanog B CORRECTION or og V5 Vector NN NNH Nanog NN NNH ect an V5 og wt -V -N WB: irA an FL FL B N WB: COS αNanog (Anti-V5) <n.s. (Anti-M2 FLAG) -+ -+ -+-+ -+-+ <NanogV5 * <FL-Nanog IP: IP: <NanogV5 FLAG <FL-Nanog Anti-V5 Anti- M2 * (Anti-Nanog) (Anti-V5) < < < < <NanogV5 < NanogV5 < (Anti-V5) Input Input < BirAV5 Fig. 1. Confirmation of Nanog–Nanog interaction (dimerization). (A) West- * * * * * ern blot analysis of gel filtration fractions containing protein complexes using * anti-Nanog antibody. Fractions that may contain Nanog homodimers are indicated with a red rectangle (Upper). A close-up of fractions containing potential Nanog dimers and monomers is also presented (Lower). Sample fractions were loaded in four (Upper) and two (Lower) denaturing polyacryl- amide gels, respectively, and Western data are presented with thin vertical lines separating the different gels. (B) CoIP in heterologous 293T cells. 293T cells were transiently transfected with constructs indicated, and total lysates were prepared and incubated with anti-M2-FL agarose (Left) or anti-V5 1 2 3 4 5 6 7 8 9 10 11 12 13 agarose (Right) overnight. Unbound material was washed away; bound ma- terial was eluted and subjected to Western blot analyses using the antibodies Fig. 4. Tethered Nanog mutants maintain DNA-binding capacity. (A) Sche- indicated (Upper). Total lysates as input were also subjected to Western blot matic depiction of the tethered Nanog dimer (NN) and monomer (NNH) analyses with anti-V5 antibody (Lower). Ͻ n.s. indicates a nonspecific signal; mutants. The 22-aa flexible polypeptide linker is shown. (B) EMSA of mutants’ * indicates FL-Nanog. The thin vertical lines indicate removal of irrelevant binding to the Cdx2 (lanes 1–9) and the Gata6 (lanes 10–13) promoter DNA lanes and juxtaposition of the two lanes relevant for this study. sequences. * indicates specific binding signals; Ͻ denotes the supershift bands by anti-Nanog. Wild-type (wt) and vector-transfected COS extracts were used as negative controls. The thin vertical line indicates removal of irrelevant lanes and juxtaposition of lanes relevant for this study. www.pnas.org PNAS ͉ June 24, 2008 ͉ vol. 105 ͉ no. 25 ͉ 8801–8802 Downloaded by guest on September 28, 2021 FLbioNanog FLbioNanog10WA A Sall4V5 BirAV5 Zfp198V5 Zfp281V5 Zfp281V5 Sall4V5 Zfp198V5 BirAV5 WB: Anti-M2 FLAG Anti-V5 IP: Anti-V5 Anti-M2 FLAG Input Anti-V5 B 10WA C D Nac1V5 Oct4V5 Nanog Nanog Nanog 10WA 10WA bio bio bio FL FL FL Nanog + + - Nanog Nanog Dax1V5 Nanog WB: bio BirAV5 bio bio --+ bio FL FL FL Anti-M2 FLAG WB: FL <n.s. Anti-Nanog * <FLbioNanog Anti-Nanog Anti-V5 IP: Anti-V5 IP: Anti-V5 Anti-V5 IP: Anti-V5 Anti-V5 Anti-M2 Anti-Nanog Anti-M2 FLAG FLAG Input Input Anti-V5 Input Anti-V5 Anti-V5 Fig. 3. Nanog homodimerization is required for its interaction with other pluripotency factors. (A) CoIP of V5his-tagged pluripotency network proteins Sall4, Zfp281, Zfp198, and BirA (control) with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. (B) CoIP of V5his-tagged Dax1 and BirA (control) with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. Ͻ n.s. indicates a nonspecific signal; * indicates the FLbioNanog signal. Note the much lower input level of FLbioNanog than FLbioNanog10WA and yet a higher IP signal (*) of FLbioNanog than FLbioNanog10WA.(C) CoIP of V5his-tagged Nac1 with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. (D) CoIP of V5his-tagged Oct4 with dimeric FLbioNanog and monomeric FLbioNanog10WA mutants. The thin vertical lines indicate removal of irrelevant lanes and juxtaposition of the two lanes relevant for this study. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804442105 8802 ͉ www.pnas.org Downloaded by guest on September 28, 2021 Requirement of Nanog dimerization for stem cell self-renewal and pluripotency Jianlong Wang*, Dana N. Levasseur*, and Stuart H. Orkin*†‡ *Division of Hematology-Oncology, Children’s Hospital and Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, and †Howard Hughes Medical Institute, Boston, MA 02115 Contributed by Stuart H. Orkin, March 6, 2008 (sent for review February 29, 2008) Pluripotency of embryonic stem (ES) cells is maintained by tran- mega-Daltons (6). By protein microsequencing we demonstrated scription factors that form a highly interconnected protein inter- that Nanog associates with numerous other critical factors that, action network surrounding the homeobox protein Nanog. En- in aggregate, form a tight protein interaction network apparently forced expression of Nanog in mouse ES (mES) cells promotes dedicated to pluripotency (6). Here, we pursue how Nanog self-renewal and alleviates their requirement for leukemia inhib- functions in this context. itory factor (LIF). Understanding molecular mechanisms by which As a divergent homeobox protein, Nanog is most closely related Nanog functions should illuminate fundamental properties of stem to mouse NK2 family members Nkx2.3 (8) and NKx2.5 (9). NKx2.5 cells and the process of cellular reprogramming. Previously, we has been studied in detail for its direct DNA contacts (18) and showed that Nanog forms multiple protein complexes in mES cells. homodimerization via the homeodomain (HD) (19). HD proteins Here, we demonstrate that Nanog dimerizes through its C-terminal often form homodimeric and heterodimeric complexes through domain rather than the homeodomain. Dimerization is required for their HDs in specifying their transcriptional actions. In some cases, interaction with other pluripotency network proteins. We also homodimerization may result in cooperative DNA-binding activity show that enforced expression of the Nanog dimer, but not the (20), whereas in other instances it may result in the inhibition of monomer, functionally replaces wild-type Nanog to sustain LIF- DNA-binding and/or transcriptional activity (21). Outside of its independent self-renewal of ES cells. Our results demonstrate that HD, Nanog shares little homology with NK2 proteins. Prior work Nanog–Nanog homodimerization is a critical aspect of its function suggests that the N-terminal domain (ND) and C-terminal domain promoting stem cell pluripotency. (CD) of mouse Nanog possess transactivator function in conven- tional reporter assays (22), whereas the CD of human Nanog may embryonic stem cells ͉ homeoprotein be functionally dominant in transactivation (23). The CD is dis- tinctive for a prominent tryptophan-rich (WR) subdomain that is highly conserved between mouse and human and acts as a strong mbryonic stem (ES) cells, derived from the inner cell mass transactivator in reporter assays (24). of the early mouse embryo (1, 2), are distinguished by E To study how Nanog functions in the pluripotency network, and unlimited self-renewal potential and the capacity for multilin- how it associates with other network proteins to regulate target eage differentiation. An understanding of the molecular under- gene expression, we have explored the structure–function relation- pinnings of these properties requires elucidation of regulatory ship of the Nanog protein in vivo in mouse ES (mES) cells. We show networks operative in ES cells. Initial efforts in dissecting that the Nanog polypeptide assembles in a homodimer that is transcriptional (3–5) and protein interaction (6) networks of mediated by the CD rather than the HD, and Nanog–Nanog mouse and/or human ES cells form a foundation for further homodimerization is necessary for its interaction with other critical mechanistic studies. factors in the pluripotency network. Finally, we provide functional Several transcription factors, notably the homeobox proteins evidence supporting a requirement of Nanog–Nanog dimerization Oct4 (7) and Nanog (8, 9), as well as the HMG box containing Sox2 in stem cell self-renewal and pluripotency. protein (10), play fundamental roles in early development and stem cell pluripotency. These key factors act in combination to sustain Results pluripotency by activating ES cell critical factors (including them- Nanog Forms Homodimers in Regulating Stem Cell Activity. To study selves) and repressing differentiation-promoting genes. ES cells are how Nanog exerts transcriptional regulation on target gene sensitive to the dosage of Nanog (11) and Oct4 (12). Enforced expression, its HD–DNA contact was modeled after mouse expression of Nanog relieves ES cells from their leukemia inhibitory NKx2.5, which predicted two possibilities: Nanog might act on factor (LIF) requirement (9) and promotes transfer of pluripotency DNA as a monomer and/or a dimer [supporting information (SI) after cell fusion (13). In contrast, overexpression of Oct4 drives Fig. S1]. These predicted structures presuppose that Nanog, like primitive endoderm differentiation (12), possibly because of direct NKx2.5, homodimerizes via its HD (19). To ascertain whether repression of the Nanog promoter by excessive Oct4 (14). In Nanog, indeed, forms homodimers in vivo, we performed size addition, Sox2 stabilizes ES cells in a pluripotent state by main- exclusion chromatography of ES cell nuclear extracts to frac- taining the requisite level of Oct4 expression (15).
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