Graded Nodal/Activin Signaling Titrates Conversion of Quantitative Phospho-Smad2 Levels into Qualitative Embryonic Stem Cell Fate Decisions Kian Leong Lee1,2*., Sandy Keat Lim2,3,4., Yuriy Lvovich Orlov2,5, Le Yau Yit1, Henry Yang6, Lay Teng Ang2, Lorenz Poellinger1,7, Bing Lim2,8* 1 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore, 2 Genome Institute of Singapore, Agency for Science, Technology, and Research, Singapore, Singapore, 3 Duke-NUS Graduate Medical School, National University of Singapore, Singapore, Singapore, 4 NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore, 5 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia, 6 Singapore Immunology Network, Agency for Science, Technology, and Research, Singapore, Singapore, 7 Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, 8 Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node, and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse embryonic stem cells leads to their exit from self-renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2, the primary downstream transcriptional factor of the Nodal/Activin pathway, followed by massively parallel sequencing, and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression. Citation: Lee KL, Lim SK, Orlov YL, Yit LY, Yang H, et al. (2011) Graded Nodal/Activin Signaling Titrates Conversion of Quantitative Phospho-Smad2 Levels into Qualitative Embryonic Stem Cell Fate Decisions. PLoS Genet 7(6): e1002130. doi:10.1371/journal.pgen.1002130 Editor: Bing Ren, Ludwig Institute for Cancer Research and University of California San Diego, United States of America Received December 13, 2010; Accepted May 1, 2011; Published June 23, 2011 Copyright: 2011 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Agency for Science, Technology, and Research (http://www.a-star.edu.sg/); Singapore National Research Foundation (http://www.nrf.gov.sg/); and Singapore Ministry of Education (http://www.moe.gov.sg/) under the Research Centre of Excellence Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: csilkl@nus.edu.sg (KLL); limbl@gis.a–star.edu.sg (BL) . These authors contributed equally to this work. Introduction Morphogens are secreted signaling molecules that orchestrate the spatial distribution and sequence of cellular differentiation events throughout embryonic development. The specific cell types, their localization and order of induction from recipient stem cell populations are determined by the concentration gradient of morphogens diffusing from the source of secretion. Previous studies have proposed some of the models by which morphogen gradients are initiated, established and stabilized including the level of receptor occupancy, positive/negative feedback and feed forward mechanisms [1–3]. However, little is understood about the transcriptional mechanisms responding to variable receptor activation and how they permit pluripotent stem cells to interpret signaling levels and direct the appropriate differentiation programs during mammalian development. Nodal and Activin are morphogens of the TGFb superfamily of signaling molecules. In Xenopus embryos, Activin is a potent concentration-dependent inducer of mesoderm, mesendoderm and endoderm in animal cap cells [2,4,5]. Nodal has also been shown to be a classical morphogen in zebrafish where it functions in a concentration gradient independently of any relaying mechanisms [6]. In the early mouse embryo, mutations that perturb the level of Nodal/Activin signaling show that the pathway plays crucial roles in the induction of the primitive streak/mesoderm, mammalian organizer (node), mesendoderm and endoderm during the establishment of the anterior-posterior axis [7–11]. In contrast to in vivo evidence that Nodal/Activin PLoS Genetics | www.plosgenetics.org 1 June 2011 | Volume 7 | Issue 6 | e1002130