SU(HW) Fine-Tunes Nuclear Lamina Interactions subset of 2001 single genes (black dots in E and F). The region around each border from which data was taken ranges from the center of the inter- LAD region to the center of the LAD; this ensures that each data point is used only once. X-axis depicts the position relative to the nearest LAD border; positive coordinates inside, negative coordinates outside LADs. (D) Median gene coverage. (E) mRNA levels in A-values, (log2(Cy5)+log2(Cy3))/ 2 (F) Median RpII18 occupancy on entire genes as determined by DamID [data from30]. doi:10.1371/journal.pone.0015013.g001 insulator protein has a unique binding pattern, and co-occurrence of different insulators is present but relatively rare (Fig. S2b). An exception is formed by DWG and BEAF-32, which exhibit substantial overlap in their binding pattern. To validate the insulator DamID profiles we first compared four of the profiles with recent chromatin immunoprecipitation (ChIP) data [31,34]. This shows that most of the insulator binding peaks detected with DamID coincide with corresponding ChIP peaks (Fig. S3a). As a second and independent validation method we compared the DamID profiles of SU(HW), CTCF and GAF with the occurrence of their cognate DNA binding motifs. The high co- occurrence of DamID binding sites with the corresponding DNA binding motifs (Fig. S3b) further validates the generated profiles. For DWG, ChIP data and a DNA binding motif have not been published before, but the overlap of the DWG and BEAF-32 binding profiles (Fig. 2a, Fig. S2b) is consistent with the reported direct interaction between DWG and BEAF-32 in vivo [35]. We conclude that we have successfully generated high-resolution genome-wide binding profiles for five insulator proteins, together with the NL interaction profile, in one and the same Drosophila cell type. SU(HW) sites are enriched at LAD borders and within LADs Next, we compared insulator binding profiles with the LAD pattern. Visual inspection led to two observations. First, SU(HW) frequently binds at multiple sites within LADs, while the other four insulator proteins mainly bind outside LADs (Fig. 2a, Fig. S2a). Second, many LAD borders coincide with a binding peak of one of the insulator proteins. To address whether these observations generally apply at a genome-wide level, we calculated the density of insulator binding peaks across the 824 aligned LAD borders (Fig. 2b). Hypothet- ically an enrichment at LAD borders or inside LADs would result in respective profiles as depicted in the first two upper panels. The profile of a protein that is depleted from LADs (which would not be expected to be directly involved in LAD formation) is expected to yield a profile as depicted in the third panel. DWG, BEAF-32, CTCF and GAF fall into this latter class: they show similar overall profiles, with the majority of binding sites occurring outside LADs. Their binding frequency gradually decreases across the LAD borders and does not show a peak at the borders themselves, indicating that these four proteins are not specifically enriched at LAD borders. For comparison, the Ecdyson Receptor (EcR) [36], a transcription factor not expected to be linked to NL interactions, yields a similar depletion from LADs. Thus, DWG, BEAF-32, CTCF and GAF are unlikely to play prominent roles in the direct regulation of LAD – NL interactions. In contrast, SU(HW) exhibits a distinct profile with two prominent regions of enrichment. First, SU(HW) binding peaks are preferentially located in the vicinity of LAD borders, with the highest frequency occurring just outside LADs, at ,4 kb from the borders. Second, the profile confirmed the visually observed enrichment of SU(HW) binding peaks inside LADs. Strikingly, within LADs the SU(HW) binding peaks are not equally distributed, but instead are concentrated at a distance of ,40 kb from the nearest LAD border (see below). This occurrence of a SU(HW) peak within the 35–45 kb region from one or the other border is found in a substantial part (51%) of the LADs of which half the size is at least 45 kb (data not shown). Performing the same alignment analysis with ChIP-defined insulator binding peaks [31] results in similar patterns, thereby confirming the validity of our findings with data from an independent experiment and technique (Fig. S3c). Further statistical analyses confirmed the enrichment of SU(HW) and the depletion of the other four proteins in LADs. For DWG, BEAF-32, CTCF and GAF respectively 4.6%, 14.3%, 17.5% and 33.7% of the binding peaks are located within LADs, while 40.6% is expected by chance. In contrast, 48.1% of the SU(HW) peaks are located within LADs, which is more than expected by chance (top panel Fig. 2c). Statistical testing against random permutation simulations showed that this enrichment of SU(HW) and the depletion of the other insulators in LADs is significant (p,1023). Furthermore, ,17% of the SU(HW) binding peaks outside LADs are located within border regions (defined as the 10 kb areas just outside LADs), which is a statistically significant enrichment (Statistical testing against random permu- tation simulations: p,1023) (bottom panel Fig. 2c). None of the four other insulator proteins are significantly enriched in LAD border regions (Statistical testing against random permutation simulations p.0.01). DWG and BEAF-32 are even significantly depleted from border regions (p,1023) while GAF and CTCF bind randomly in LAD border regions, as would be expected by chance. In total, 77% of the LADs and 27% of the LAD border regions contain a least one SU(HW) binding peak. To address whether SU(HW) is specifically enriched inside LADs and not just in repressive chromatin in general, we compared the SU(HW) binding peaks to Polycomb-bound regions (Fig. S4), which are large chromatin domains that are mostly transcriptionally inactive [37] and only partly overlap with LADs. The amount of overlap between Polycomb domains and LADs is roughly as may be expected by random chance, namely 40%. Statistical analysis revealed that, in contrast to LADs, Polycomb domains are significantly depleted of SU(HW) binding sites. We find 4.9% of the SU(HW) peaks to be located within Polycomb domains, while 9.3% is expected by chance, showing that the enrichment of SU(HW) is specific for NL-interacting chromatin. Taken together, these results demonstrate that SU(HW) binding is significantly and specifically enriched just outside LAD borders, as well as at specific locations within LADs (red triangles in Fig. 2d). Furthermore, even though DWG, BEAF-32, dCTCF and GAF binding sites occasionally overlap with individual LAD borders, they do not show a statistically significant global preference for LADs or LAD borders (colored triangles in Fig. 2d). Enrichment of SU(HW) is sequence driven The observed enrichment of SU(HW) and the paucity of the other four insulator proteins in LADs could be dictated by the genomic distribution of the corresponding DNA binding motifs, which is likely since the presence of a SU(HW) DNA binding motif is known to be highly predictive for SU(HW) binding [38]. Alternatively, the patterns could be driven by respectively cooperative or exclusive interactions with other chromatin compo- nents in LADs. To discriminate between these two mechanisms we compared the occurrence of protein binding peaks to the distribution of the corresponding sequence motifs for CTCF, GAF and SU(HW) (respectively grey and colored lines in Fig. 3a). PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e15013