Understanding the mechanism by which embryonic stem (ES) cells self-renew is crucial for the realization of their therapeutic potential. Earlier, overexpression of Id proteins was shown to be sufficient to maintain mouse ES cells in a self-renewing state even in the absence of serum. Here, we use ES cells derived from Id deficient mice to investigate the requirement for Id proteins in maintaining ES cell self-renewal. We find that Id1−/− ES cells have a defect in self-renewal and a propensity to differentiate. We observe that chronic or acute loss of Id1 leads to a down-regulation of Nanog, a critical regulator of self-renewal. In addition, in the absence of Id1, ES cells express elevated levels of Brachyury, a marker of mesendoderm differentiation. We find that loss of both Nanog and Id1 is required for the up-regulation of Brachyury, and ectopic Nanog expression in Id1−/− ES cells rescues the self-renewal defect, indicating that Nanog is the major downstream target of Id1. These results identify Id1 as a critical factor in the maintenance of ES cell self-renewal and suggest a plausible mechanism for its control of lineage commitment.
Get full access to this article
View all access options for this article.
References
1.
MartinGR. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A, 78:7634–7638.
2.
EvansMJ, KaufmanMH. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature, 292:154–156.
3.
SmithAG. 2001. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol, 17:435–462.
KimJ, ChuJ, ShenX, WangJ, OrkinSH. 2008. An extended transcriptional network for pluripotency of embryonic stem cells. Cell, 132:1049–1061.
6.
WangJ, RaoS, ChuJ, ShenX, LevasseurDN, TheunissenTW, OrkinSH. 2006. A protein interaction network for pluripotency of embryonic stem cells. Nature, 444:364–368.
7.
LohYH, WuQ, ChewJL, VegaVB, ZhangW, ChenX, BourqueG, GeorgeJ, LeongB. et al. 2006. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet, 38:431–440.
8.
ChenX, XuH, YuanP, FangF, HussM, VegaVB, WongE, OrlovYL, ZhangWet al. 2008. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell, 133:1106–1117.
9.
MitsuiK, TokuzawaY, ItohH, SegawaK, MurakamiM, TakahashiK, MaruyamaM, MaedaM, YamanakaS. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 113:631–642.
10.
ChambersI, ColbyD, RobertsonM, NicholsJ, LeeS, TweedieS, SmithA. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 113:643–655.
11.
HoughSR, ClementsI, WelchPJ, WiederholtKA. 2006. Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog. Stem Cells, 24:1467–1475.
12.
IvanovaN, DobrinR, LuR, KotenkoI, LevorseJ, DeCosteC, SchaferX, LunY, LemischkaIR. 2006. Dissecting self-renewal in stem cells with RNA interference. Nature, 442:533–538.
13.
KurodaT, TadaM, KubotaH, KimuraH, HatanoSY, SuemoriH, NakatsujiN, TadaT. 2005. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol Cell Biol, 25:2475–2485.
PerrySS, ZhaoY, NieL, CochraneSW, HuangZ, SunXH. 2007. Id1, but not Id3, directs long-term repopulating hematopoietic stem-cell maintenance. Blood, 110:2351–2360.
22.
CochraneSW, ZhaoY, WelnerRS, SunXH. 2009. Balance between Id and E proteins regulates myeloid versus lymphoid lineage decisions. Blood, 113:1016–1026.
23.
YingQL, NicholsJ, ChambersI, SmithA. 2003. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell, 115:281–292.
24.
WilkinsonDG, BhattS, HerrmannBG. 1990. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature, 343:657–659.
25.
SuzukiA, RayaA, KawakamiY, MoritaM, MatsuiT, NakashimaK, GageFH, Rodriguez-EstebanC, Izpisua BelmonteJC. 2006. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc Natl Acad Sci U S A, 103:10294–10299.
26.
SuzukiA, RayaA, KawakamiY, MoritaM, MatsuiT, NakashimaK, GageFH, Rodriguez-EstebanC, BelmonteJC. 2006. Maintenance of embryonic stem cell pluripotency by Nanog-mediated reversal of mesoderm specification. Nat Clin Pract Cardiovasc Med, 3,Suppl 1:S114–S122.
JamesD, NoggleSA, SwigutT, BrivanlouAH. 2006. Contribution of human embryonic stem cells to mouse blastocysts. Dev Biol, 295:90–102.
29.
BrennandK, HuangfuD, MeltonD. 2007. All beta cells contribute equally to Islet growth and maintenance. PLoS Biol, 5:e163.
30.
GaoD, NolanDJ, MellickAS, BambinoK, McDonnellK, MittalV. 2008. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science, 319:195–198.
31.
MoffatJ, GruenebergDA, YangX, KimSY, KloepferAM, HinkleG, PiqaniB, EisenhaureTM, LuoBet al. 2006. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell, 124:1283–1298.
32.
RozenS, SkaletskyH. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol, 132:365–386.
ZhuangY, GudasLJ. 2008. Overexpression of COUP-TF1 in murine embryonic stem cells reduces retinoic acid-associated growth arrest and increases extraembryonic endoderm gene expression. Differentiation, 76:760–771.
35.
JenY, WeintraubH, BenezraR. 1992. Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins. Genes Dev, 6:1466–1479.
36.
YanW, YoungAZ, SoaresVC, KelleyR, BenezraR, ZhuangY. 1997. High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice. Mol Cell Biol, 17:7317–7327.
37.
PanL, SatoS, FrederickJP, SunXH, ZhuangY. 1999. Impaired immune responses and B-cell proliferation in mice lacking the Id3 gene. Mol Cell Biol, 19:5969–5980.
38.
HayashiK, LopesSM, TangF, SuraniMA. 2008. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell, 3:391–401.
39.
LimLS, LohYH, ZhangW, LiY, ChenX, WangY, BakreM, NgHH, StantonLW. 2007. Zic3 is required for maintenance of pluripotency in embryonic stem cells. Mol Biol Cell, 18:1348–1358.
40.
FariaTN, LaRosaGJ, WilenE, LiaoJ, GudasLJ. 1998. Characterization of genes which exhibit reduced expression during the retinoic acid-induced differentiation of F9 teratocarcinoma cells: involvement of cyclin D3 in RA-mediated growth arrest. Mol Cell Endocrinol, 143:155–166.
41.
MasuiS, OhtsukaS, YagiR, TakahashiK, KoMS, NiwaH. 2008. Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells. BMC Dev Biol, 8:45.
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
