KRAB–Zinc Finger Proteins and KAP1 Can Mediate Long-Range Transcriptional Repression through Heterochromatin Spreading Anna C. Groner 1,2 , Sylvain Meylan 1,2 , Angela Ciuffi 3 , Nadine Zangger 1,2 , Giovanna Ambrosini 1,4 , Nicolas De ´ nervaud 1 , Philipp Bucher 1,4 , Didier Trono 1,2 * 1 School of Life Sciences, E ´ cole Polytechnique Fe ´de ´rale de Lausanne (EPFL), Lausanne, Switzerland, 2 Frontiers-in-Genetics National Center of Competence in Research, E ´ cole Polytechnique Fe ´de ´rale de Lausanne (EPFL), Lausanne, Switzerland, 3 Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland, 4 Swiss Institute of Bioinformatics, Lausanne, Switzerland Abstract Kru ¨ ppel-associated box domain-zinc finger proteins (KRAB–ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB–mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB–containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB–mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 b (HP1b) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1–dependent transcriptional repression at an endogenous KRAB–ZFP gene cluster, where KAP1 binds to the 39 end of genes and mediates propagation of H3K9me3 and HP1b towards their 59 end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB–ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB–ZFPs and KAP1. Citation: Groner AC, Meylan S, Ciuffi A, Zangger N, Ambrosini G, et al. (2010) KRAB–Zinc Finger Proteins and KAP1 Can Mediate Long-Range Transcriptional Repression through Heterochromatin Spreadin. PLoS Genet 6(3): e1000869. doi:10.1371/journal.pgen.1000869 Editor: Hiten D. Madhani, University of California San Francisco, United States of America Received October 20, 2009; Accepted February 2, 2010; Published March 5, 2010 Copyright: ß 2010 Groner 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 supported by grants from the Swiss National Science Foundation, the Infectigen Association, and the European Union. 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: didier.trono@epfl.ch Introduction The proper control of gene expression is paramount to all cellular events, and is orchestrated through a sophisticated balance of activating and repressing influences. Kru ¨ ppel-associated box domain zinc finger proteins (KRAB-ZFP) constitute the single largest group of transcriptional repressors encoded by the genomes of higher organisms. After appearing in early tetrapods, the KRAB-ZFP family has rapidly expanded and diverged through multiple rounds of gene and segmental duplications, to give rise to more than three hundred and fifty members annotated in both mice and humans [1–6]. In spite of their numerical abundance, wide range of tissue-specific expression and dynamic evolutionary history, the physiological functions of KRAB-ZFPs collectively remain ill-defined, and few of their targets have been identified [3,7]. However, emerging evidence links KRAB/KAP1-mediated regulation to processes as essential and diverse as stem cell pluripotency, early embryonic development and differentiation, genomic imprinting, response to DNA damage and control of behavioral stress [8–14]. Furthermore, KAP1 controls endogenous retroviruses in embryonic stem cells, a process crucial for the maintenance of genomic stability [15]. KRAB-ZFPs all harbor a so-called KRAB domain situated upstream of an array of two to forty C2H2 zinc fingers, which provide sequence-specific DNA binding ability [2]. KRAB recruits KAP1 (KRAB-associated protein 1, also known as TRIM28, Tif1b or KRIP-1) [16–19], which acts as a scaffold for various heterochromatin-inducing factors, such as heterochromatin protein 1 (HP1), the histone methyltransferase SETDB1, the nucleosome-remodeling and histone deacetylation (NuRD) com- plex, the nuclear receptor corepressor complex 1 (N-CoR1) and, at least during early embryonic development, de novo DNA methyltransferases [20–27]. The phylogenetically conserved family of HP1 proteins is implicated in a variety of nuclear events, such as transcriptional repression and maintenance of chromosome structure [28]. HP1 harbors two major regions: the chromo domain, which binds to repressive di- and trimethylated histone 3 lysine 9 (H3K9me2 and H3K9me3, respectively) residues, and the chromo shadow domain, involved in HP1 homodimerization and recruitment of PLoS Genetics | www.plosgenetics.org 1 March 2010 | Volume 6 | Issue 3 | e1000869