itself, showing the enrichment just outside LAD borders as well as at +40 kb. Taken together, the enrichment of SU(HW) at LAD borders as well as at 40 kb inside LADs is confirmed by the binding of CP190, and thus is likely to involve functional insulator protein complexes. SU(HW) binding antagonizes genome - NL interactions The surprising pattern of SU(HW) binding relative to LADs suggested two possible roles for SU(HW) in the regulation of genome - NL associations. First, the binding of SU(HW) at LAD borders could help to separate LADs from inter-LADs. Second, the SU(HW) binding inside LADs could modulate NL interactions of LADs. To directly test these hypotheses, we monitored the genome-wide changes in NL interactions after alteration of the expression level of SU(HW). Specifically, we either reduced SU(HW) levels by RNA interference (RNAi), or we increased SU(HW) levels by transfection with a SU(HW) expression vector. We then created new full-genome DamID maps of NL interactions. For RNAi we used two different, non-overlapping, double- stranded RNA (dsRNA) fragments to exclude off-target effects. Treatment with a dsRNA fragment derived from the unrelated white gene served as a control. Western blot analysis showed that both su(Hw) dsRNA fragments caused efficient knockdown of the SU(HW) protein (Fig. 4a, 1st panel). Knockdown of SU(HW) had no effect on the doubling time of the cells (data not shown), ruling out secondary effects of an altered cell cycle on the DamID pattern. Elevated levels of SU(HW) were obtained by co- transfection of the DamID plasmids with a vector that drives expression of SU(HW) from an Act5C promoter. Western blot analysis showed only a slight increase in expression of SU(HW), presumably because only a minority of cells is transfected (Fig. 4a, 2nd panel). However, because the overexpression vector is co- transfected with the DamID vector, overexpression may be expected to be more prominent in cells that express Dam-LAM. We generated DamID maps of NL association for each treatment in two independent experiments. If SU(HW) is involved in the demarcation of LAD borders, we reasoned that loss of SU(HW) would lead to changes in NL interactions near the borders where SU(HW) is bound, such as expansion or contraction of LADs, or decreased sharpness of borders. However, the median NL interaction profile across LAD borders with SU(HW) bound within 10 kb outside the LAD showed no dramatic change at the borders: the median curve did not shift laterally, nor did it change in steepness when comparing SU(HW) knockdown to control cells (blue vs grey in Fig. 4b). Overexpression of SU(HW) also had no detectable effect on LAD border sharpness or position (orange vs grey in Fig. 4c). We therefore conclude that under the conditions and in the cell type tested, SU(HW) is not essential for the demarcation of LAD borders, despite the clear enrichment of SU(HW) binding sites at LAD borders. In contrast, we did notice effects of altered SU(HW) levels on genome - NL interactions inside LADs. Knockdown of SU(HW) caused a specific increase in NL interaction levels inside LADs, while overexpression of SU(HW) had the opposite effect inside LADs (color vs grey in the 1st panels of Fig. 4b,c). Thus, SU(HW) antagonizes genome – NL interactions inside LADs. To confirm the specificity of this effect, we repeated this analysis after dividing LADs into three classes: LADs without SU(HW) binding peaks inside, LADs with at least one SU(HW) binding peak inside and the 25% of LADs with the highest SU(HW) peak density (Fig. 5a,b). This shows that the effects of altered SU(HW) SU(HW) Fine-Tunes Nuclear Lamina Interactions expression levels are restricted to LADs that harbor SU(HW) binding sites, and that the overall effect is proportional to the density of SU(HW) peaks. To visualize this dependency further we calculated the average change in genome – NL interactions per LAD (Fig. 5c), showing that the change in NL interactions is indeed increasing with higher SU(HW) peak density. A Wilcoxon test between the grouped LADs confirmed NL interactions to significantly increase and decrease after respectively knockdown and overexpression of SU(HW) (p,1023). Repeating this analysis for the individual replicates showed the same trend (Fig. S6), thereby confirming the reproducibility of the observed changes in genome – NL interactions. Together, these results demonstrate that SU(HW) reduces the frequency of NL interactions inside LADs. The observation that only LADs with SU(HW) binding sites are affected by changes in SU(HW) levels indicates that SU(HW) controls genome – NL interactions locally rather than globally. To investigate the range over which SU(HW) acts in cis, we plotted the change in NL interactions as a function of the distance to the nearest SU(HW) binding site, after knockdown and overexpression of SU(HW) (Fig. 5d). This reveals that the antagonistic effect of SU(HW) on NL interactions is most pronounced within ,5 kb from the SU(HW) binding sites, but extends to .10 kb. A weaker effect is also seen at SU(HW) sites in border regions and outside LADs, indicating that in these regions SU(HW) may help to suppress spurious contacts with the NL. Interestingly, the range of the SU(HW) effects on NL interactions corresponds approximately to the width of the SU(HW) binding peaks (Fig. 5e). Moreover, an elevated baseline of SU(HW) interactions, extending over .10 kb from the binding peak centers, is present specifically inside LADs. Thus, the cis-effect of SU(HW) on NL interactions has a similar distribution as the actual contacts of SU(HW) with sequences surrounding the SU(HW) binding sites. Taken together, our data reveal that SU(HW) binding antagonizes genome - NL interac- tions in a local fashion, over a distance that roughly corresponds to the range over which SU(HW) contacts the genome. Discussion Here, we report the fine structure of LADs in a Drosophila cell line, and analyze the genome-wide distribution of five insulator proteins with respect to LADs. The DamID method is particularly suited for the detailed mapping of NL interactions. DamID offers molecular resolution that cannot be obtained by traditional microscopy-based methods such as fluorescent in situ hybridiza- tion. This allowed us to compare NL interactions and insulator protein binding sites at a resolution of ,1 kb. We discovered that SU(HW) is the only tested insulator protein that preferentially interacts with DNA in LADs and at LAD borders. Furthermore, we demonstrate that SU(HW) modulates genome – nuclear lamina (NL) interactions by local antagonism inside LADs. Because interactions as detected by DamID require at least transient physical contact of the Dam-Lam protein with the chromatin fiber, we interpret changes in DamID signals as local changes in the molecular contact frequency between the NL and the probed genomic locus. SU(HW) as an antagonist of LAD – NL interactions The found enrichment of SU(HW) in LADs supports previous ideas about the peripheral localization of SU(HW). SU(HW) is thought to form large aggregates that are often located at the nuclear periphery [42] and a SU(HW) protein complex member, TOPORS, is shown to interact with the NL. The association with PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e15013