play a key role in chromatin organization by mediating intra- and interchromosomal interactions. They have been shown to block the communication between a promoter and its enhancer when placed in between [19,20,reviewed in 21] and some insulators are thought to separate inactive from active chromatin domains by acting as a barrier against spreading of repressive chromatin proteins or histone modifications into neighbouring regions [reviewed in 21,22]. In Drosophila five main insulator proteins have been identified; Suppressor of Hairy-wing (SU(HW)), CCCTC-binding Factor (CTCF), Boundary Element-associated Factor (BEAF-32), Zeste- white 5 (Zw5) -also known as Deformed Wings (DWG)- and GAGA Factor (GAF) [19,23,24,25,26,27,reviewed in 28]. Espe- cially SU(HW) is a promising candidate to regulate genome – NL interactions since the SU(HW) complex member TOPORS is shown to interact with lamin proteins [29]. Here we analyze the possible roles of insulator proteins in the regulation of NL- genome interactions in Drosophila Kc cells. We report a high resolution map of Drosophila genome – NL interactions, showing that Drosophila LADs exhibit remarkably similar characteristics as their human counterparts. Comparison to new and published binding maps for all five insulators revealed SU(HW) to be the only insulator protein that preferentially binds at LAD borders and at specific positions inside LADs. By direct functional studies we demonstrate that SU(HW) modulates LAD – NL interactions through a local antagonistic effect. We thus identified SU(HW) as the first protein to fine-tune molecular interactions between the genome and the NL. Results LADs in the Drosophila genome A previous DamID study in Drosophila Kc cells identified hundreds of genes that associate with the NL [7]. However, this study lacked the resolution required for a detailed view of genome - NL interaction patterns. We therefore repeated these DamID experiments for LAM (also known als Lamin-Dm0, the only B- type lamin in Drosophila), this time using a high-density microarray that queried the entire fly genome with a median probe spacing of ,300 bp. With DamID, DNA adenine methyltransferase (Dam) fused to LAM leaves a stable adenine-methylation ‘footprint’ in vivo at the interaction sites. Previous comparisons to fluoresence in situ hybridization data have indicated that DamID signals obtained with LAM can be interpreted as relative molecular contact frequencies between the NL and the probed genomic locus [7,8]. Note that the Dam-LAM fusion protein is expressed at very low levels, preventing overexpression artifacts. We averaged the data of two independent DamID experiments, which highly correlated with each other (Pearson correlation of 0.77) and with the previously published low resolution data (Pearson correlation of 0.74). The resulting profile (Fig. 1a, Fig. S1) shows that the genome in Kc cells is associated with the NL through large continuous domains, alternating with regions of low association. A domain detection algorithm, previously developed for the analysis of human NL interaction data [8] identified a total of 412 Drosophila Lamina Associated Domains (LADs) (Table S1). These LADs vary in size between 7 and 700 kb, with a median size of ,90 kb (red line in Fig. 1b). In total they cover 40% of the genome (data not shown). The Drosophila genome is much more compact than the human genome. Although the overall gene counts differ by only 2-fold (respectively 14,449 and ,31,000 genes), the Drosophila genome is ,25 times smaller, and Drosophila genes are typically shorter and more closely spaced than human genes. These differences in scale raise the question whether the organization of the Drosophila SU(HW) Fine-Tunes Nuclear Lamina Interactions genome in LADs also occurs at a smaller scale. Indeed, human LADs are much bigger, ranging from ,0.1 Mb to ,10 Mb with a median LAD size of 553 kb (blue line in Fig. 1b) [8]. However, we find that the number of genes per LAD is remarkably similar between the two species, on average respectively 6.8 and 8.5 genes/LAD in human and Drosophila cells (Fig. 1c). These observations suggest that LAD organization has co-evolved with the linear spacing and size of genes, and that the number of genes, rather than the absolute length of DNA, is an important structural parameter of LADs. Taken together, these results demonstrate that the organization of the genome into LADs is conserved between Drosophila and human cells, albeit at different scales. Drosophila LADs are repressed chromatin domains Human LADs represent a repressive chromatin environment with a relatively low gene density. To investigate whether Drosophila LADs exhibit similar characteristics, we aligned the 412 LADs by their left as well as their mirrored right borders and calculated the average profiles for several features across the 824 combined borders. This analysis revealed that 45–55% of the sequence within LADs consists of genes, while outside LADs the gene coverage is ,70% (Fig. 1d). In total, the 412 LADs in Kc cells contain about 30% of all genes. To assess the expression status of these genes we measured the mRNA expression levels of nearly all genes in Kc cells using microarrays and calculated the expression profile across the aligned LADs. As is the case for human LADs, almost all genes inside LADs are expressed at baseline levels, while genes outside LADs display varying and on average higher expression levels (Fig. 1e). Consistent with this, the binding of the 18-kDa subunit of RNA polymerase (RpII18) to genes [30] shows a low median level and a low variance inside LADs, compared to inter-LAD regions (Fig. 1f). The median mRNA expression levels and RpII18 binding levels both exhibit a sharp transition at LAD borders (red lines in Fig. 1e and 1f), similar to what has been reported for human LADs [8]. Taken together, Drosophila LADs exhibit similar characteristics as their human counterparts: they represent a repressive type of chromatin with sharp transitions. Genome-wide identification of in vivo binding sites of insulator proteins Next, we investigated whether specific insulator proteins are involved in regulating LAD formation. Such an activity requires the candidate insulator protein to bind either inside LADs or at LAD borders. We therefore conducted DamID experiments in Kc cells to obtain whole-genome binding maps for the five known Drosophila insulator proteins: BEAF-32, CTCF, DWG, GAF and SU(HW). Full-genome binding profiles of DWG and GAF were previously not available for Drosophila Kc cells. Although for CTCF, SU(HW) and BEAF-32 such maps have been reported [31], a proper comparison requires that all the insulator profiles are obtained from the same cells and under the same experimental conditions. For each insulator protein we performed two independent DamID experiments, which highly correlated with each other (Pearson correlation coefficients between 0.71 and 0.83) and with previously published low resolution DamID data (Pearson correlation coefficients of 0.64 for GAF [32] and 0.70 for SU(HW) and BEAF32 [33]). We averaged the duplicate datasets to obtain a single full-genome profile for each protein. The resulting binding profiles of all five insulator proteins are generally characterized by sharp peaks of local enrichment (Fig. 2a, Fig. S2a). A peak detection algorithm (see Methods) identified 2,173 peaks for DWG; 4,027 for BEAF-32; 1,290 for GAF; 2,930 for CTCF; and 2,986 for SU(HW) within the Drosophila genome. Each PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e15013