Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation Yunzhe Zhang1,2., Marissa Cooke2,3., Shiraj Panjwani1,2., Kaixiang Cao1,2, Beth Krauth3, Po-Yi Ho1,2, Magdalena Medrzycki1,2, Dawit T. Berhe2, Chenyi Pan1,2, Todd C. McDevitt2,3, Yuhong Fan1,2* 1 School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 2 The Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 3 The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America Abstract Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes. Citation: Zhang Y, Cooke M, Panjwani S, Cao K, Krauth B, et al. (2012) Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation. PLoS Genet 8(5): e1002691. doi:10.1371/journal.pgen.1002691 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America Received September 2, 2011; Accepted March 21, 2012; Published May 10, 2012 Copyright: 2012 Zhang 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 is supported by a Johnson & Johnson/GA Tech Healthcare Innovation Award (to YF and TCM), a Georgia Cancer Coalition Distinguished Scholar Award (to YF), NIH grant GM085261 (to YF), NSF EBICS Science and Technology Center (CBET-0939511), the Georgia Tech Integrative BioSystems Institute, and Georgia Institute of Technology. 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: Yuhong.fan@biology.gatech.edu . These authors contributed equally to this work. Introduction Pluripotent embryonic stem cells (ESCs) can self-renew and differentiate into diverse cell types, including lineages from all three germ layers present in the adult organism, offering great promise in regenerative medicine in addition to serving as a useful system for developmental biology studies. The epigenome and transcriptional circuitry of pluripotent stem cells have been extensively investigated, and chromatin and epigenetic signatures have emerged as key components in defining and regulating stem cell pluripotency [1–4]. Recent reports have associated ESCs with a particularly open, hyperdynamic chromatin and hyperactive global transcription [2,5,6], and open chromatin has been suggested as a marker for pluripotency [7,8]. However, it remains undetermined whether higher order chromatin compaction is required for pluripotent stem cell differentiation and how an open chromatin state impacts stem cell function. In eukaryotic cells, histones are the major structural proteins that associate with DNA to form chromatin. The basic repeating unit of chromatin is the nucleosome core particle, which consists of an octamer of four core histones (H2A, H2B, H3 and H4) wrapped by 146 bp of DNA [9]. Further compaction of chromatin into higher order structures, such as a 30 nm fiber, is facilitated by binding of H1 linker histones to DNA entry/exit points of nucleosomes and linker DNA between nucleosomes. Reducing the total amount of H1 in vivo leads to a relaxed chromatin structure [10–12]. The H1 histone family is the most divergent and heterogenous group of histones among the highly conserved family of histone proteins. In mammals, 11 non-allelic H1 subtypes have been identified, including five somatic H1 subtypes (H1a–e), the replacement subtype H10, four germ cell specific H1 subtypes (oocyte specific H1oo, and testis-specific H1t, H1t2, H1LS1) as well as a more recently identified and distantly related subtype H1x [13]. Although the individual depletion of each of the three major somatic H1 subtypes, H1c, H1d and H1e, in mice does not lead to any detectable changes in total H1 levels or obvious phenotypes [14], deletion of H1c, H1d and H1e altogether leads to nearly a 50% reduction of total H1 levels and embryonic lethality with a broad phenotype [15], demonstrating that critical levels of total H1 histones are essential for mouse embryogenesis. PLoS Genetics | www.plosgenetics.org 1 May 2012 | Volume 8 | Issue 5 | e1002691