SU(HW) Fine-Tunes Nuclear Lamina Interactions Figure 3. SU(HW) distribution relative to LADs is sequence driven and linked to CP190 binding. (A) Profiles of sequence motifs across aligned LAD borders. X-axis depicts the position relative to the nearest LAD border; positive coordinates inside LADs and negative coordinates outside LADs. Colored lines show the median frequency within a running window of 10 kb for sequence motifs, grey lines for DamID identified peaks. (B) Profiles of CP190 peaks [28] across aligned LAD borders; all Cp190 peaks (1st panel), CP190 peaks without SU(HW) binding, defined as peaks with an average log2(SU(HW)binding ratio) ,1.0 (2nd panel), CP190 peaks with SU(HW) binding, defined as CP190 peaks with an average log2(SU(HW)binding ratio) .1.5 (3rd panel). doi:10.1371/journal.pone.0015013.g003 The enrichment of SU(HW) at +40 kb can not be explained by a subgroup of specifically sized LADs, since alignments of subgroups of differently sized LADs all result in an enrichment at ,40 kb (data not shown). The enrichment of SU(HW) at +40 kb is also not caused by a genome-wide periodicity of SU(HW) binding itself: a histogram of the pair-wise distances between all SU(HW) peaks shows no preferential spacing of SU(HW) peaks in the range of 40 kb (Fig. S5). In addition, we found no significant correlation between the presence of SU(HW) at a LAD border and binding of SU(HW) in the same LAD around +40 kb (Fisher’s exact test p.0.1, data not shown). Thus, SU(HW) shows preferences for LAD borders as well as for the +40 kb position within LADs, but these SU(HW) binding events appear not to be linked. In summary, these results show that the remarkable pattern of SU(HW) relative to LADs is driven by the distribution of its binding motif in the genome. Enrichment of SU(HW) containing CP190 peaks indicates functionality. To further investigate the remarkable pattern of SU(HW) relative to LADs we analysed the distribution across aligned LAD borders of CP190 binding peaks (defined with ChIP by Bushey et al. [31]). CP190, a subunit of different insulator protein complexes, is thought to be necessary for insulator function since it is essential for both insulator body formation and enhancer blocking activity [31,39,40,41]. Figure 3b (1st panel) shows that CP190 is mostly bound outside LADs. Interestingly, the profile also exhibits a modest local peak of enrichment inside LADs, exactly at ,40 kb from the LAD borders. CP190 insulator complexes can be divided in at least three different subclasses, containing either SU(HW), CTCF or BEAF-32 [31]. The profile of CP190 binding peaks across LAD borders can therefore be subdivided in peaks that co-localize with SU(HW) and peaks that do not (Figure 3b, 2nd and 3rd panels). Remarkably, CP190 binding peaks that do not contain SU(HW) binding (defined as CP190 peaks with an average log2(SU(HW)binding ratio) ,1) are depleted from LADs, without any enrichment at +40 kb. They probably represent the BEAF-32 and CTCF containing subclasses of CP190 binding peaks. Strikingly, the Su(Hw) containing subclass (defined as CP190 peaks with an average log2(SU(HW)- binding ratio) .1.5) strongly resembles the profile of SU(HW) PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e15013