Cohesin Is Required for Higher-Order Chromatin Conformation at the Imprinted IGF2-H19 Locus Raffaella Nativio1., Kerstin S. Wendt2.¤, Yoko Ito1, Joanna E. Huddleston1, Santiago Uribe-Lewis1, Kathryn Woodfine1, Christel Krueger3, Wolf Reik3,4, Jan-Michael Peters2"*, Adele Murrell1"* 1 Department of Oncology, University of Cambridge, Cancer Research UK Cambridge Research Institute, Cambridge, United Kingdom, 2 Institute of Molecular Pathology, Vienna, Austria, 3 Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge, United Kingdom, 4 Centre for Trophoblast Research, University of Cambridge, Cambridge, United Kingdom Abstract Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi–mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF–mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin’s function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion. Citation: Nativio R, Wendt KS, Ito Y, Huddleston JE, Uribe-Lewis S, et al. (2009) Cohesin Is Required for Higher-Order Chromatin Conformation at the Imprinted IGF2-H19 Locus. PLoS Genet 5(11): e1000739. doi:10.1371/journal.pgen.1000739 Editor: Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom Received May 20, 2009; Accepted October 23, 2009; Published November 26, 2009 Copyright: 2009 Nativio 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: Funding for this work was from Cancer Research UK Senior Cancer Research Fellowship and Association for International Cancer Research Project grant (AM), as well as the support of The University of Cambridge, Cancer Research UK, and Hutchison Whampoa Limited (AM, RN, YI, JEH KW, SU-L). WR and CK are funded by EMBO, DFG, BBSRC, MRC, and the EU NoE The Epigenome. Research in the laboratory of J-MP is supported by Boehringer Ingelheim and the Austrian Science Fund via the special research program ‘‘Chromosome Dynamics.’’ 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: Jan-Michael.Peters@imp.ac.at (J-MP); amm207@cam.ac.uk (AM) ¤ Current address: Department of Cell Biology and Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands . These authors contributed equally to this work. " These authors are joint senior authors on this work. Introduction Cohesin is an evolutionarily conserved protein complex composed of the core subunits, SMC1, SMC3, SCC1/RAD21 and SCC3/SA (reviewed in [1]). It has been proposed that cohesin mediates sister chromatid cohesion by embracing replicated DNA molecules as a ring [2]. Cohesin also has important roles in gene regulation in yeast, animals and humans (reviewed in [1,3]). This regulatory function also exists during G1 phase and in post-mitotic cells, indicating that cohesin affects gene expression independent of its role in cohesion [4–6]. Cohesin mediates gene regulation at least in part by interaction with insulator elements [6–11]. Insulators are chromatin bound- aries that separate gene promoters from regulatory elements. The only known protein that directly binds insulators in mammalian cells is the multi-functional zinc finger protein CTCF (CCCTC binding factor, reviewed in [12]). Several studies have recently identified co-localisation of CTCF and cohesin in mammalian genomes and have shown that CTCF is needed to recruit cohesin to these binding sites [6,9–11]. Remarkably, although CTCF can associate with its binding sites in the absence of cohesin, its enhancer blocking activity depends on cohesin [6,10]. It has, therefore, been speculated that CTCF may mediate transcription- al insulation by recruiting cohesin to particular sites in the genome [6], but it remains unknown how cohesin controls gene regulation at these sites. The IGF2-H19 locus plays a role in the aetiology of embryonic growth disorders and in various cancers (reviewed in [13]). A CTCF mediated insulator sequence plays a role in the reciprocal imprinting of IGF2 and H19 genes [14–16]. This insulator is located upstream of the H19 gene and is known as the imprinting control region (ICR). It acquires methylation on the paternal allele during male germ cell development and is therefore also called the H19 differentially methylated region (DMR) or domain (DMD). CTCF binding at the insulator prevents the IGF2 gene from accessing enhancers downstream of the H19 gene. This physical PLoS Genetics | www.plosgenetics.org 1 November 2009 | Volume 5 | Issue 11 | e1000739