The Insulator Protein SU(HW) Fine-Tunes Nuclear Lamina Interactions of the Drosophila Genome Joke G. van Bemmel1, Ludo Pagie1, Ulrich Braunschweig1, Wim Brugman3, Wouter Meuleman1,2,4, Ron M. Kerkhoven3, Bas van Steensel1* 1 Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands, 2 Division of Molecular Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands, 3 Central Microarray Facility, Netherlands Cancer Institute, Amsterdam, The Netherlands, 4 Delft Bioinformatics Lab, Delft University of Technology, Delft, The Netherlands Abstract Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs) in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that the Drosophila genome is also organized in discrete LADs, which are about five times smaller than human LADs but contain on average a similar number of genes. Systematic comparison to new and published insulator binding maps shows that only SU(HW) binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW) weakens genome – NL interactions through a local antagonistic effect, but we did not obtain evidence that it is essential for border formation. Our results provide insights into the evolution of LAD organization and identify SU(HW) as a fine-tuner of genome – NL interactions. Citation: van Bemmel JG, Pagie L, Braunschweig U, Brugman W, Meuleman W, et al. (2010) The Insulator Protein SU(HW) Fine-Tunes Nuclear Lamina Interactions of the Drosophila Genome. PLoS ONE 5(11): e15013. doi:10.1371/journal.pone.0015013 Editor: Gert Jan C. Veenstra, Radboud University Nijmegen, The Netherlands Received August 25, 2010; Accepted October 5, 2010; Published November 24, 2010 Copyright: 2010 van Bemmel 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 financially supported by the Netherlands Genomics Initiative and a European Young Investigator Award to BvS. 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: b.v.steensel@nki.nl Introduction The nuclear lamina (NL), a dense fibrillar network covering the inside of the nuclear membrane in metazoan cells [reviewed in 1], is thought to represent a major structural element for the nuclear organization of the genome. Close contacts between the NL and chromatin have been observed by electron microscopy [2] and more recently by three-dimensional structured illumination microscopy [3]. Based on FISH studies specific loci are known to preferentially localize at the periphery [reviewed in 4,5]. Genome-wide mapping using the DamID technology [6] in Drosophila Kc cells demonstrated hundreds of genes to be in molecular contact with the NL [7]. These genes are strongly repressed and lack active histone marks. Application of the same mapping technology at a higher resolution in human lung fibroblasts showed that NL interactions occur through large continuous genomic domains with sharply defined borders [8]. In these Lamina Associated Domains (LADs) gene expression is strongly repressed, RNA Polymerase II (RNApolII) and active histone marks are depleted, and repressive histone marks are enriched. Several observations indicate that LADs are not just passively pushed towards the periphery, but instead are the result of specific NL – genome interactions. Human LAD borders tend to be marked by sequence elements such as outward orientated promoters, CTCF binding sites and CpG islands [8], which indicates that the association with the NL could be controlled by DNA sequence. Furthermore, loss or mutation of lamins in flies and mammals can cause dissociation from the periphery and changes in gene expression, histone modifications and binding of chromatin proteins [9,10,11,12,13,14] indicating the functional relevance of genome – lamina associations. In addition, upon differentiation hundreds of genes move from or towards the NL, correlating with their respectively increased and decreased expression levels [7,15,16]. Taken together these point to the existence of mechanism that regulate genome-NL interactions. The current knowledge about such a regulatory mechanism is limited. Repressive histone marks are most likely not involved, since loss of histone methylatransferases or DNA methylatrans- ferases do not effect peripheral localization of single loci [15,17]. Possibly the absence of active histone marks could play a role, since treatment with an HDAC inhibitor has been shown to disrupt molecular interactions with the NL in Drosophila Kc cells [7] and to cause dissociation of genes from the nuclear periphery in mammalian cells [18]. Alternatively, DNA-binding proteins that physically interact with the NL could be involved in modulating NL interactions. Because a subset of human LAD borders is marked by an insulator element, the CTCF binding sequence [8], we reasoned that insulator proteins are likely candidates to be involved in modulating genome-NL interactions. Insulator elements are DNA sequences that, when bound by insulator proteins, are thought to PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15013