Title The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. BMYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity. Citation: Zhan M, Riordon DR, Yan B, Tarasova YS, Bruweleit S, et al. (2012) The B-MYB Transcriptional Network Guides Cell Cycle Progression and Fate Decisions to Sustain Self-Renewal and the Identity of Pluripotent Stem Cells. PLoS ONE 7(8): e42350. doi:10.1371/journal.pone. 0042350 Editor: Qiang Wu, National University of Singapore, Singapore Received March 5, 2012; Accepted July 4, 2012; Published August 24, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This research was supported in by the Intramural Research Program of the NIH, National Institute on Aging, and by a scholarship (SB) from Boehringer Ingelheim Fonds, Foundation for Basic Research in Medicine (Heidesheim, Germany). 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: bohelerk@grc.nia. nih.gov ¤a Current address: The Methodist Hospital Research Institute, Cornell University Weill Cornell Medical College, Houston, Texas, United States of America ¤b Current address: Hong Kong Baptist University, Hong Kong, China