The role of Polycomb in stem cell genome architecture Gloria Mas 1,2 and Luciano Di Croce 1,2,3 Polycomb-group proteins maintain embryonic stem cell identity by repressing genes that encode for developmental regulatory factors. Failure to properly control developmental transcription programs by Polycomb proteins is linked to disease and embryonic lethality. Recent technological advances have revealed that developmentally repressed genes tend to cluster in the three-dimensional space of the nucleus. Importantly, spatial clustering of developmental genes is fundamental for the correct regulation of gene expression during early development. Here, we outline novel insights and perspectives regarding the function of Polycomb complexes in shaping the stem cell genome architecture, and discuss how this function might be required to properly orchestrate transcriptional programs during differentiation. Addresses 1 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain 2 Universitat Pompeu Fabra (UPF), Barcelona, Spain 3 ICREA, Pg. Lluis Companys 23, 08010 Barcelona, Spain Corresponding authors: Mas, Gloria (gloria.mas@crg.eu) and Di Croce, Luciano (luciano.dicroce@crg.eu) Current Opinion in Cell Biology 2016, 43:87–95 This review comes from a themed issue on Differentiation and disease Edited by Tom Misteli and Graham Warren http://dx.doi.org/10.1016/j.ceb.2016.09.006 0955-0674/# 2016 Elsevier Ltd. All rights reserved. Introduction During development, pluripotent embryonic stem cells (ESCs) differentiate into all the specialized cell types of an embryo. At every stage of embryogenesis, a precise gene expression program determines the identity of each cell. The gene expression programs that dictate cell identity are orchestrated by a multilayered molecular system that includes transcription factors, epigenetic modifiers, and chromatin remodelers [1]. Recent advances in high-resolution microscopy and chromosome conformation capture (3C) technologies have revealed that the three-dimensional organization of the genome also plays a critical role in transcriptional regulation [2]. A growing body of research argues that chromosome folding within the nucleus brings distant regions of the genome into close proximity, and that this physical interaction influences their function. On the large scale, chromo- somes occupy discrete sections of the nuclear space known as chromosome territories (reviewed in [3]) (Figure 1). Within chromosome territories, the genome is folded into topologically associating domains (TADs). TADs are megabase-sized regions comprised of highly interacting genomic loci that are flanked by structural boundaries and stabilized by architectural proteins, such as CTCF and cohesin (Figure 1). Genomic regions within a TAD tend to share the same epigenetic and transcrip- tional features (reviewed in [2,3]). At increasing resolu- tion, the physical contacts between distal regulatory sequences (‘enhancers’) and gene promoters form chro- matin loops (Figure 1). Chromatin loops are mediated by protein effectors, non-coding RNAs, and histone post- translational modifications (PTMs) [1,4,5]. Importantly, large-scale organization of the genomes in TAD struc- tures is a highly conserved feature across mammalian cell types and species [6,7]. However, local dynamic topolog- ical changes can occur at the level of chromatin loops, and these have been proposed to influence gene transcription and ensure the proper orchestration of cellular differenti- ation [4,8  ,9]. Polycomb-group (PcG) proteins are key regulators of the transcriptional programs that maintain stem cell proper- ties and dictate lineage specification [10–12]. In mouse ESCs, PcG proteins encompass two enzymatically dis- tinct complexes, the Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). PRC1 comprises one of the E3 ligases Ring1A/B, which catalyze the mono-ubiquitina- tion of histone H2A at lysine 119 (H2AK119ub1), as well as one each of the Pcgf and Phc proteins and either a Cbx protein or RYBP. PRC2 contains the core subunits Ezh1/ 2, Suz12 and Eed, and catalyzes methylation of lysine 27 of histone H3 (H3K27me3). Association of PcG pro- teins at selected genomic loci maintains transcriptional gene repression. Gene silencing is achieved by direct inhibition of the transcription machinery and/or by pre- venting chromatin accessibility to remodeling complexes. Hence, in ESCs, Polycomb complexes maintain lineage- specific genes in a silenced state and prevent exit of cells from pluripotency. Here, we review the latest research on the consequence of Polycomb loss in genome topology at the level of a whole chromosome (for instance, the X chromosome), TADs, Polycomb domains and chromatin loops (promoter–pro- moter and promoter–enhancer interactions) (Figure 1) (Box 1). Available online at www.sciencedirect.com ScienceDirect www.sciencedirect.com Current Opinion in Cell Biology 2016, 43:87–95