Modular Insulators: Genome Wide Search for Composite CTCF/Thyroid Hormone Receptor Binding Sites Oliver Weth, Christine Weth, Marek Bartkuhn, Joerg Leers, Florian Uhle¤, Rainer Renkawitz* Institute for Genetics, Justus-Liebig-University Giessen, Giessen, Germany Abstract The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, including insulation and enhancer blocking. We had previously identified two composite elements consisting of a CTCF and a TR binding site at the chicken lysozyme and the human c-myc genes. Using these it has been demonstrated that thyroid hormone mediates the relief of enhancer blocking even though CTCF remains bound to its binding site. Here we wished to determine whether CTCF and TR combined sites are representative of a general feature of the genome, and whether such sites are functional in regulating enhancer blocking. Genome wide analysis revealed that about 18% of the CTCF regions harbored at least one of the four different palindromic or repeated sequence arrangements typical for the binding of TR homodimers or TR/RXR heterodimers. Functional analysis of 10 different composite elements of thyroid hormone responsive genes was performed using episomal constructs. The episomal system allowed recapitulating CTCF mediated enhancer blocking function to be dependent on poly (ADP)-ribose modification and to mediate histone deacetylation. Furthermore, thyroid hormone sensitive enhancer blocking could be shown for one of these new composite elements. Remarkably, not only did the regulation of enhancer blocking require functional TR binding, but also the basal enhancer blocking activity of CTCF was dependent on the binding of the unliganded TR. Thus, a number of composite CTCF/TR binding sites may represent a subset of other modular CTCF composite sites, such as groups of multiple CTCF sites or of CTCF/Oct4, CTCF/Kaiso or CTCF/ Yy1 combinations. Citation: Weth O, Weth C, Bartkuhn M, Leers J, Uhle F, et al. (2010) Modular Insulators: Genome Wide Search for Composite CTCF/Thyroid Hormone Receptor Binding Sites. PLoS ONE 5(4): e10119. doi:10.1371/journal.pone.0010119 Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France Received September 10, 2009; Accepted March 18, 2010; Published April 9, 2010 Copyright: 2010 Weth 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 DFG. 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: rainer.renkawitz@gen.bio.uni-giessen.de ¤ Current address: Department of Anaesthesiology, University of Giessen-Marburg, Giessen, Germany Introduction The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, such as gene activation [1], gene repression [2,3] and enhancer blocking [4–13]. In vertebrates, CTCF is the only identified protein that is able to bind to insulators and to mediate enhancer blocking [4]. Insulators block the action of enhancers when positioned between enhancer and promoter, thereby preventing inappropriate action of enhancers on neigh- bouring genes. The CTCF-mediated enhancer blocking can be constitutive, for example in the locus control region of the b-globin genes [4]. In other cases, regulation of enhancer blocking has been described at the level of DNA-binding. It has been shown that CTCF is not able to bind to those binding-sites that include methylated CpGs. A well characterized system for such a regulation is the imprinting control region (ICR) of the Igf2/H19 locus. At this lCR the binding-site for CTCF on the paternal allele is methylated. This leads to inhibition of DNA-binding of CTCF and thereby to the relief of enhancer blocking [5–7,9,14–17]. Another mechanism in the regulation of DNA binding of CTCF is in the context of transcription, when RNA polymerase transcribes through the CTCF binding site. This activity dislocates CTCF from the DNA [18]. Previously, we had identified a different type of regulation of enhancer blocking. This involves thyroid hormone (T3), which can regulate enhancer blocking by CTCF [8]. A subset of CTCF binding sites is found next to thyroid hormone response elements, such as in the composite elements located in the rat element 144, the mouse c-myc gene and the human APP gene. The thyroid hormone receptor (TR) is a member of the nuclear hormone receptor family of transcription factors. TR associates with proteins that possess histone acetyltransferase activity (HAT) in the presence of its ligand T3. In the absence of T3, TR is complexed with enzymes that mediate histone deacetylation. For the composite element consisting of a CTCF and a TR binding site within the chicken lysozyme upstream silencer, CTCF binds to footprint 1 (F1), and TR binds as a homodimer or heterodimer with the retinoid-X-receptor (RXR) to footprint 2 (F2). For this element it has been demonstrated that T3 mediates relief of enhancer blocking and that activation occurs even though CTCF remains bound to its binding site [8]. Furthermore, ChIP analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter [8]. Nuclear hormone receptor binding sites next to important CTCF sites at the Igf2/H19 locus have been found, but no functional effects with respect to insulation, enhancer blocking or imprinting could be shown [19]. Here we wanted to determine whether CTCF and TR combined sites are representative of a general feature of the PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10119