Figure 1.
RNA interference (RNAi) identifies 78 factors inducing insulator reporter gene activity including NURF and dREAM components.
(A) Workflow of the RNAi screen in 66×384-well plates from the DRSC. Knockdown of 13900 genes was done with Drosophila S2 cells with the integrated F8OF8L insulator reporter construct (F8, Fab-8; O, OpIE2 enhancer; L, luciferase). (B) Top GO-terms (determined via GeneCodis [58]–[60]) for the 78 identified genes. (C) High-throughput data shown in a dotplot diagram. Z-scores are indicated for every well (well number). For many gene products several wells contain different dsRNA sequences targeting the same gene. Z-scores higher than two are highlighted in red. (D) Individual depletion of NURF and dREAM components and associated factors verify enhancer blocking function. S2 cell pools with the integrated F8OF8L insulator reporter (dark grey) or the control F8OL reporter construct (light grey) were incubated with dsRNA against factors of the NURF-complex (pink): ISWI, NURF-38, CAF1/p55, NURF301, Pzg, DREF or against the dREAM-complex (blue): CAF1/p55, Mip40, Mip130, E2F2. Reporter gene activity is expressed as fold change relative to control knockdown. Error bars indicate the standard deviation of three individual replicates. (p-values: *≤0.05, **≤0.01, ***≤0.001).
Figure 2.
Purification of either CTCF or CP190 reveals NURF and dREAM binding to both insulator factors.
(A) Interaction heatmap based on Mascot scores (dCTCF) or fold enrichment of normalized intensities (CP190), depicting associated factors identified by mass spectrometry after immunopurification of FLAG-dCTCF or FLAG-CP190 expressed in S2 cells. (B) Nuclear extracts from S2 cells (lanes 1–2, 7–10) and S2 cells stably expressing FLAG-CP190 (lanes 3–4) or FLAG-dCTCF (lanes 5–6) were precipitated with FLAG antibody (lanes 2, 4, 6), CP190 antibody (lane 9), dCTCF antibody (lane 10) or IgG (lane 8) as control. Antibodies used in Western blot are indicated on the right. Lanes 1, 3, 5 and 7: 1% Input.
Figure 3.
NURF and dREAM components co-localize with CP190 genome-wide.
(A) Correlation analysis for genome-wide binding of CP190 with 215 profiles from S2 cells (modENCODE) and 5 profiles from Kc cells [33]. Shown are the top 30 ranking factors. Components of the NURF complex are marked in pink and of the dREAM complex in blue. (B) Cluster heat map of 6,000 genomic regions with CP190 and/or CTCF sites compared with binding sites for components of NURF (NURF301, ISWI, Chro) and dREAM (E2F2, Lin-52, Mip120, Mip130, Myb) complexes. Each lane represents an 8 kb region. Scale represents binding (red) to no binding (blue).
Figure 4.
NURF and dREAM components co-localize with dCTCF/CP190.
ChIP in S2 cells with antibodies against CTCF and CP190 and components of the NURF complex (ISWI, NURF301, Pzg and Chro) and dREAM complex (Mip40, Mip120, Mip130, E2F2) or CAF-1/p55. The genomic regions tested are indicated (compare Table S2) and grouped into CTCF plus CP190, low CTCF plus CP190, low CTCF without CP190 and neither CTCF nor CP190. Error bars indicate the standard deviation of three independent experiments.
Figure 5.
Recruitment of NURF and dREAM is dependent on dCTCF and CP190 at specific sites.
ChIP in S2 cells treated with dsRNA against dCTCF and CP190 (dsCTCF/dsCP190; dark colors) or against luciferase as control (dsLuci; light colors). Antibodies were used specific for dCTCF, CP190 and components of the NURF (ISWI, Chro) and dREAM complex (Mip40, Mip120, Mip130) or, as part of both complexes, CAF-1/p55. Error bars indicate the standard deviation of three independent experiments. (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).
Figure 6.
Insulator specific effects of NURF and dREAM components.
S2 cells pools with the integrated luciferase reporter constructs with different CTCF/CP190 binding sites located between the enhancer (O, OpIE2) and the promoter of the reporter gene (L, luciferase). (A) Luciferase activity after control knockdown of GFP shows the enhancer blocking activity of Fab-8 (F8), bicoid (bcd), CG31472 and Fab-6 (F6(2)), when compared to the CTCF binding site mutant (F8mut) (top). Error bars indicate the standard deviation of three biological replicates. The different insulator reporter constructs are depicted (bottom), the genomic fragments used are indicated in Table S2 and genome browser views are in Figure S7. (B) Knockdown experiments against CTCF, ISWI or NURF301 (top) and of CTCF, CAF1/p55 or triple-knockdown of Mip-factors (bottom). Fold change of luciferase activity is calculated relative to the control knockdown. Error bars indicate the standard error of three or more individual replicates (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).
Figure 7.
NURF binding causes nucleosomal depletion at CP190 binding sites.
(A) Cumulative representation of changes in H3-binding and MNase-protection as detected by H3 ChIP-seq and MNase-seq after depletion of CTCF/CP190 (green) or ISWI (orange). Data is shown as coverage for specific knock-down normalized to luciferase control knock-down (luci) after log2-transformation. Average effects are shown across CP190 binding sites (colored) or control sites shifted 25 kb (grey). (B) All sites with increased H3 binding after CTCF/CP190 depletion (responders) show a similar H3 increase upon ISWI depletion. Non-responding sites after CTCF/CP190 depletion (non-responders) do not respond to ISWI depletion. H3 ChIP in S2 cells treated with dsRNA against CTCF and CP190 (dsCTCF/CP190; green), ISWI (dsISWI; orange) or against luciferase as control (dsLuci; black). Error bars indicate the standard deviation of two independent experiments (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).