Analysis v2.0). To correct for nonspecific binding of Dam and local differences in DNA accessibility, methylated fragments of Kc cells transfected with a Dam-only construct were labeled with a different fluorescent dye. 13 mg of labeled Dam-fusion and 13 mg of Dam-only methylated fragments were pooled and hybridized to microarrays carrying 380,000 60-mer DNA oligonucleotides [55] (Roche-NimbleGen). Median probe spacing is 300 bp. For each profile, material from two independent experiments was hybrid- ized in opposite dye orientations over Dam controls. The obtained Dam-fusion/Dam-only ratio reflects the extent of protein binding to each fragment on the microarray, corrected for local differences in chromatin accessibility. Probes are mapped to Drosophila melanogaster genome sequence release 4.3. Knockdown and overexpression of SU(HW) NL interaction profiles after knockdown of SU(HW) were obtained by using dsRNAs directed against white and su(Hw). dsRNAs were in vitro transcribed using the RiboMax kit (Promega) from PCR amplicons. PCR amplicons were designed according to the Harvard Drosophila RNAi Screening Centre (www.flyrnai.org; su(Hw) HFA17074 and MRC020_B05), or as published before for white [56]. RNAi treatments were performed as described before [30], with the exception that the treatment was repeated at day 5 and cells were transfected with DamID constructs on day 7. NL interaction profiles after overexpression of SU(HW) were obtained by co-transfection of DamID vectors and respective overexpression vectors. For overexpression of SU(HW) we used vector pAsu(Hw)iresR. As a control we used the vector pAWiresR. Genomic DNA was isolated after 48 h instead of 24 h. Expression levels of SU(HW) were monitored with Western blot analysis, presenting the protein expression level within the entire cell population. However, because typically 20–30% of cells are transfected, this yields an underestimate of the degree of overexpression. SU(HW) antibody The antibody against the C-terminal peptide of SU(HW) was kindly provided by P. Geyer [57]. Expression analysis Total RNA was extracted with TRIzol (Invitrogen) and treated with DNaseI. Isolated RNA from three independent cell cultures was labeled with Cy5 and with Cy3 and co-hybridized to INDAC oligo arrays (http://www.indac.net) printed at the NKI Central Microarray Facility, with each oligonucleotide spotted twice. Raw data from three biological replicates were loess normalized per subarray, and averaged. A-values, (log2(Cy5)+log2(Cy3))/2, were used for further analysis. Data analysis Microarray data analysis was performed with R [58]. Raw data from two biological replicates were loess normalized, median centered, and dye swap arrays were averaged. For the NL interaction profile after SU(HW) knockdown, the normalized data from the different dsRNA amplicons were averaged as well. To calculate the correlation with previously published low resolution data, the high resolution data were re-sampled to the resolution of the published cDNA microarrays by averaging values for probes from the high resolution array whose center falls within the space of one probe of the cDNA array. LADs were defined as described in [8]. In short; sharp transitions in the DamID signal were identified using a sliding edge filter (window size 199 probes), and adjacent transitions SU(HW) Fine-Tunes Nuclear Lamina Interactions exceeding a threshold (here 0.3) were combined into domains if at least 70% of the enclosed probes have a positive log2 ratio. Polycomb domains were taken from [37] and transposed to FlyBase release 4. Insulator peak positions were determined as follows: after applying a running mean of 5 probes, the derivative was calculated over the running-mean with a 7 probe window. In addition FDR-corrected p-values were determined for each probe using linear modeling (LiMMA) [59]. Peaks were assigned at transitions of the derivative from a positive to a negative value (indicating a peak) and where in addition at least three probes were significantly enriched (p,0.005). Motif scans were performed using the TFBS Perl module [60] with position weight matrices (PWMs) obtained from literature [38,61] and the TRANSFAC database [62]. Briefly, PWMs were compared against the genomic sequence and a relative matching score was calculated based on a PWM’s information content. A matching score of 85% (CTCF, SU(HW)) and 99% (GAF) was used as it yielded a similar number of matches to the identified in vivo binding sites. Custom R scripts were used to align data to LAD borders or to SU(HW) peaks; for this purpose, genome-wide positions of all analyzed features were converted to coordinates relative to the nearest border or peak. In case of LADs, data around right-side borders were mirrored and combined with data around left-side borders. The region around borders from which data was taken ranges from the middle of inter-LAD regions to the middle of LADs themselves; this ensures that all datapoints are used only once. Similarly, in case of alignments to Su(HW) peaks, the region ranged halfway to the next peak. Median binding ratios across the aligned borders or peaks were calculated with a running window covering 5% of the data within the aligned region for alignments at LADs (Fig. 1def, Fig. 4bc, Fig. 5ab), 20% and 10% for changes in NL interaction (Fig. 5c) and 10% for aligning at Su(Hw) peaks (Fig. 5d,e). Data availability DamID and expression data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE20313. Supporting Information Figure S1 Genome – NL interaction map in Drosophila Kc cells on all chromosomes. Y-axes depict the log2 transformed Dam-LAM over Dam-only methylation ratio with a running median of 15 probes. Red rectangles represent LADs. Figure S2 Insulator protein binding map in Drosophila Kc cells (A) Insulator protein binding maps at four arbitrarily chromosomal regions. Y-axes depict the Dam-insulator over Dam- only methylation ratio. Grey rectangles represent LADs. (B) Co- occurrence of insulator proteins indicated by a density plot of the log2 transformed binding ratio of each insulator protein (colored lines) at the binding peaks of each insulator protein (different panels). Figure S3 DamID maps are consistent with ChIP and sequence motif distributions. (A) Binding maps of BEAF-32, GAF, CTCF and SU(HW) at random regions of chromosome 2L for Dam-insulator over Dam-only methylation ratios (colored lines) versus ChIP scores (black). (B) DamID binding maps of CTCF and SU(HW) (colored lines) at chromosome 2L versus the location of corresponding sequence motifs (black). Figure S4 No SU(HW) enrichment in Polycomb do- mains. (A) Binding maps of insulator proteins along a five sequential 1Mb regions at chromosome 2L. Y-axes depict the linear Dam-SU(HW) over Dam-only methylation ratio. Grey rectangles represent the Polycomb domains. PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e15013