SU(HW) Fine-Tunes Nuclear Lamina Interactions Figure 5. SU(HW) is a local antagonist of genome – NL interactions. (A–B) Median NL interaction (log2 Dam-LAM/Dam ratio) across LADs without SU(HW) peaks (190 borders, 1st panel), LADs with at least one SU(HW) peak (634 borders, 2nd panel, red triangle represents the presence of one or more SU(HW) peaks at any position inside LADs), the 25% of LADs with the highest SU(HW) peak density (206 borders, 3rd panel, red triangles represent high density of SU(HW) peaks). (A) After knockdown of SU(HW) (blue line), after control knockdown (grey line). (B) After overexpression of SU(HW) (orange line), after control overexpression (grey line). (C) Ave changes in NL interaction levels per LAD, for LADs without SU(HW) (grey), LADs with at least one SU(HW) peak (light blue or orange), the 25% of LADs with the highest SU(HW) peak density (dark blue or orange) after knockdown (blue, 1st panel) and overexpression of SU(HW) (orange, 2nd panel). * Wilcoxon test; p,1023 (D) Median changes in NL interaction across aligned SU(HW) peaks (red triangle) inside LADs (bright lines), in border regions (pale lines) and outside LADs (grey lines) after knockdown of SU(HW) (blue, 1st panel) and after overexpression of SU(HW) (orange, 2nd panel). (E) Cis-spreading of SU(HW) DamID signals from aligned SU(HW) binding peaks (red triangle), inside LADs (red line) and outside LADs (grey line). doi:10.1371/journal.pone.0015013.g005 have been suggested to interact with each other [44,45] to form chromatin loops in a SU(HW) dependent manner [46]. Hence, if SU(HW) promotes associations between a LAD and a locus located in the nuclear interior, then naturally the LAD would be less frequently located at the NL. The presence of a protein inside LADs that antagonizes LAD – NL interactions is somewhat paradoxical. We propose that SU(HW) is important for the fine-tuning of NL interactions. For example, by loosening of LAD – NL associations, SU(HW) may facilitate switches in NL associations of some loci during cellular differentiation. In this respect it is interesting to note that SU(HW) is expressed at particularly high levels in embryos and pupae (http://flybase.org/reports/FBgn0003567.html), when many cells differentiate into new cell types, while the expression is low in larvae and adult flies when relatively few cells change their identity. In addition a fraction of the SU(HW) binding sites has been found to be cell-type specific [31]. Although the antagonistic effect of SU(HW) appears relatively modest at individual loci, the large numbers of SU(HW) sites in many LADs may together modulate chromosome organization to a significant degree. Evolutionary aspects of LADs and their borders Our high-resolution map of NL interactions reveals that the fly genome is organized into hundreds of discrete LADs, similar to the human genome. Like in human LADs, most genes in fly LADs exhibit low occupancy by the RNA polymerase II 18 kD subunit (RPII18) and they are expressed at low levels. Even though Drosophila LADs are about five-fold smaller than human LADs, the number of genes per LAD is remarkably similar between the two species. This suggests that LAD organization has co-evolved with the linear spacing and size of genes along the two genomes. Human and fly LADs are also similar in their demarcation of borders by specific insulator proteins. Surprisingly, the two species employ different insulators for this purpose. In human fibroblasts, a substantial amount of LAD borders is marked by CTCF. In Drosophila cells, we observed no such enrichment of CTCF; instead SU(HW) is enriched at LAD borders. SU(HW) is an insect–specific protein [47]. The switch of insulator protein utilization at LAD borders between the two species is remarkable, because this involves not only a functional switch of the two proteins, but simultaneously the co- evolution of the respective binding motifs at many LAD borders. While the enrichment of SU(HW) near LAD borders is clearly non-random and sequence-based, we were unable to detect significant consequences of the loss or overexpression of SU(HW) on genome – NL interactions around LAD borders. Since SU(HW) alone is not essential for demarcation of LAD borders, we suggest that SU(HW) is redundant with one or more of its partner proteins, such as CP190 or Mod(mdg4) [39,48]. It is also possible that SU(HW) is only important at LAD borders in specific cell types. Effects on gene regulation? Given the strong overall repression of genes in LADs, together with observations that the NL can actively contribute to gene silencing [14,49,50], it may be expected that the effects of SU(HW) on genome – NL interactions also have impact on gene repression in LADs. Interestingly, gypsy elements, which harbor strong SU(HW) binding sites, can boost transgene expression at many integration sites [51]. Possibly the antagonistic action of SU(HW) on NL interactions contributes to this anti-silencing effect. However, analysis of microarray expression data after SU(HW) knockdown or overexpression in Kc cells (data not shown) did not reveal preferential misregulation of genes in SU(HW)-marked LADs or genes that have respectively increased or decreased levels of NL interactions. Redundant mechanisms may provide robust- ness to gene repression in LADs and may thus mask a contribution of SU(HW). This notion is supported by the fact that su(Hw) mutant flies are viable and exhibit a phenotype that is restricted to impaired oogenesis (Phenotypic Descriptions of Classical Alleles from http://flybase.org/reports/FBgn0003567.html). Finally, it should be considered that the effects of SU(HW) on genome – NL interactions, and thus on the spatial organization of interphase chromosomes, may be important for other nuclear processes, such as the regulation of DNA replication or repair [52]. Materials and Methods Constructs Dam-LAM (pDamMyc-Dm0), Dam-Gaf (pNDamMyc-Gaf) and control Dam-only (pNDamMyc) constructs have been described previously [6,7,34]. To obtain pGWNDamMyc-su(Hw), pGWNDamMyc-CTCF, and pGWC-Dwg-MycDam, the open reading frames were amplified from cDNA clones (LD15893, GH14774, LD44361, LD45751) and cloned in-frame with Dam by using TOPO cloning and GATEWAY recombination as described [30]. Dam-su(Hw) and Dam-Beaf32 were published before [33]. For overexpression of SU(HW) we constructed vector pA- su(Hw)-iresR, and as a control pAWiresR. To obtain pAWir- esRFP, the internal ribosome entry site (IRES) from pUAST- 3xEGFP [53] was amplified using primers that incorporate a STOP codon just 59 of the IRES, and SacI sites at both ends of the amplicon. This fragment was cloned into the SacI site of pAWR (The Drosophila Gatewaya Vector Collection, a resource developed by the Murphy lab, Carnegie Institute) to yield pAWiresR. pA- su(Hw)-iresR was obtained by GATEWAY recombination with pAWiresR. DamID DamID was performed as described before [54]. In brief, Dam- fusion and Dam-only expression vectors were transfected in parallel into separate dishes of Kc cells by electroporation. Genomic DNA was isolated after 24 h and adenine-methylated fragments were amplified from genomic DNA by methylation- specific PCR. 1 mg of amplified methylated DNA was labeled with Cy-dye labeled random nonamers (TriLink Biotechnologies, according to NimbleChip Arrays User’s Guide: ChIP-chip PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e15013