NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization

Abstract

Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.

Figure 1.

NURF301 affects the formation of gypsy insulator bodies. (A) Localization of insulator bodies is disrupted after depletion of individual gypsy insulator proteins. Right panels are quantification of average total GFP foci number per cell and average number of large foci (volume > 0.5 μm³) per cell imaged on glass slides. Cells were grown at 25°C. (B) Workflow of high-content RNAi imaging screen: Pre-spot dsRNA on plates, dispense cell suspension, incubate 4 days at RT then 2.5 h at 4°C, fix and DAPI stain, image, and process data. (C) Average large foci number per nucleus after each dsRNA treatment. Z’ factor = 0.8, indicating a good assay. (D) Verification on glass slides that NURF301 knockdown can alter the formation of gypsy insulator bodies. Cells were grown at 25°C, and these samples were processed in parallel with cells in (A). Quantification of the average total foci and large foci number are shown on the right. Scale bars: 5 μm. Comparisons in groups are all compared with mcherry dsRNA knockdown control cells. Three biological replicates were performed with n > 500 cells examined in each condition. Two-tailed unpaired t-test was used, and error bars show standard deviation. **P < 0.01 and ^****P < 0.0001.

Figure 2.

NURF301 promotes gypsy insulator barrier activity. (A) Schematic diagram of in vivo insulator barrier assay. In the non-insulated UAS-luciferase line, spreading of repressive chromatin leads to low expression of luciferase. In the insulated line, presence of the gypsy insulator acts as a barrier and allows for high expression of luciferase. (B) Western blotting of male second instar larval extracts for NURF301, CP190, and Tubulin (loading control) in control flies and NURF301^RNAi knockdown flies using the Act5C-Gal4 driver grown at 25°C. (C, D). Relative luciferase activity of insulated or non-insulated male (C) and female (D) third instar larvae of control or with NURF301^RNAi driven by Mef2-Gal4 driver. Su(Hw)^RNAi is used as a positive control. For each genotype, n = 12 individual larvae, with each experiment performed twice with similar results. One-way ANOVA followed by Tukey HSD post hoc test was used to calculate P-values for pairwise comparisons. Box represents the 25–75th percentiles, and the median is indicated. The whiskers show minimum and maximum values. P-values are indicated.

Figure 3.

NURF301 interacts physically with gypsy insulator proteins, and these factors co-localize genome-wide. (A) Co-immunoprecipitation of NURF301 with core gypsy components. Nuclear extracts (NE) are from embryos aged 0–24 h collected at RT, which is the same material used for IP followed by mass spectrometry. NE was immunoprecipitated with each antibody or with normal serum (IgG) as indicated. Unbound supernatant (Sup) and bound (IP) fractions are also shown. Polycomb (Pc) is used as a negative control. (B) Representative screenshot of ChIP-seq profiles shows NURF301 co-localizes extensively with gypsy core components in Kc cells grown at 25°C. Peaks called by MACS2 are indicated by black bars. Three biological replicates were examined for each sample. (C) Binary heatmap of CP190, NURF301, Su(Hw), and Mod(mdg4)67.2 binding sites in Kc cells, ordered by supervised hierarchical clustering. Each column represents a single independent genomic location, and a mark in a row indicates presence of the indicated factor. Total peaks of each factor are shown on the right. 7423 genomic sites co-localize between NURF301 and CP190, and 1624 genomic sites (yellow bar) were bound by all four factors. (D) Average signals of CP190 and NURF301 peaks, but not of Su(Hw) and Mod(mdg4)67.2, accumulate at the TSS.

Figure 4.

Depletion of NURF301 decreases the binding of gypsy core components throughout the genome. (A) Screenshot of ChIP-seq profiles of CP190, Su(Hw), and NURF301 in control cells and Nurf301 knockdown cells. Cells were grown at 25°C. Peaks in control samples are indicated by black rectangles, and decreased peaks determined by the Diffbind algorithm (FDR < 0.05) in knockdown samples are indicated by blue rectangles. Three biological replicates were performed for each condition. (B) Su(Hw) and CP190 ChIP-seq signal at called peaks centered on their summit, which are sorted by descending signal in control cells. Left panel is Su(Hw), right panel is CP190 divided into three groups based on whether it overlaps with Su(Hw) (group I), is located near TAD borders (group II), and remaining sites (group III). Corresponding signals are shown for control cells (left) or after NURF301 or Su(Hw) depletion (right). (C) Binary heatmap of CP190, Su(Hw), and their respective decreased peaks after NURF301 depletion. At 708 genomic sites, both CP190 and Su(Hw) are decreased in Nurf301 knockdown cells. (D) Total and differential peaks of Su(Hw) and CP190 in three groups in (B) after depletion of NURF301. (E) Differentially bound CP190 and Su(Hw) ChIP-seq peaks in Nurf301 knockdown were validated by ChIP-qPCR. Chromatin association of CP190 and Su(Hw) at Girdin, CR43718, and Ptth was reduced after depletion of NURF301. Normal serum is used as a negative control for ChIP, and CG4896 is used as a negative control site, at which binding of CP190 and Su(Hw) are not affected by NURF301. Data are from two independent biological replicates and measured using four technical replicates for each sample. The p-values were calculated using Student's t-test. ns: not significant, * P < 0.05, **P < 0.01.

Figure 5.

Su(Hw) assists the recruitment of NURF301 at a subset of sites. (A) Representative screenshot of decreased NURF301 after knockdown of Nurf301, su(Hw), or Cp190 in Kc cells. Cells were grown at 25°C. Peaks called in control samples are indicated by black rectangles, and decreased peaks in knockdown conditions are shown as blue rectangles. Three biological replicates were performed for each condition. (B) ChIP-seq signals for NURF301 are classified into three groups based on overlap with Su(Hw) (group I), location near TAD borders (group II), and remaining sites (group III) in control and Su(Hw)- or CP190-depleted cells. (C) Motif enrichment of decreased NURF301 peaks after depletion of Su(Hw). (D) Binary heatmap of NURF301 and decreased NURF301 after depletion of Su(Hw) or CP190. (E) Protein levels of NURF301, CP190, Su(Hw), and Mod(mdg4)67.2 in total cell lysate, cytoplasmic and soluble nuclear fraction, and chromatin-bound fractions of control and knockdown cells, as indicated. LaminB and Tubulin were blotted as a control for a chromatin-bound and cytoplasmic protein, respectively. Quantification of NURF301 protein level is graphed at the bottom. Data are from four biological replicates and paired t-test was used. *P < 0.05, **P < 0.01.

Figure 6.

NURF301 specifically affects the co-localization of insulator bodies with gypsy insulator DNA binding sites in the nucleus. (A) Screenshot of the ChIP-seq profiles of CP190/Su(Hw)/Mod(mdg4)67.2/NURF301 in 30 kb probe regions. Black bars represent peaks identified by MACS2 in control Kc cells. Pink and blue rectangles indicate increased and decreased peaks in knockdown samples, respectively. Probe A was used as a control, which has no CP190/Su(Hw)/Mod(mdg4)67.2 colocalized sites (gypsy sites). Probe B has multiple sites with enriched signals of CP190/Su(Hw)/Mod(mdg4)67.2. (B) Representative images of CP190 immunostaining and Oligopaint FISH signals in Kc cells grown at 25°C after dsRNA treatment as indicated. Scale bar represents 2 μm. (C) Percentage of cells displaying contact between CP190 and respective probes. Data are from five biological replicates, each dot represents one biological replicate, n > 300 cells per replicate. (D) For cells showing contact in (C), average overlapped volume between CP190 and probes relative to CP190 volume. Overlap value was normalized to the volume of CP190 to exclude the bias of increased size of insulator bodies in Nurf301 and su(Hw) knockdown cells. Paired t-test was used, and error bars show standard deviation. ns, not significant; *P < 0.05 as indicated.

Figure 7.

Depletion of NURF301 specifically alters 3D arrangement of gypsy insulator binding sites. (A) Schematic of gypsy probes on Chr3L. The co-occupied sites of Su(Hw)/CP190/Mod(mdg4)67.2 are designated as gypsyF probe, and the 1D reverse of non-gypsy sites are designated as control probe (gypsyR). The DNA amount labeled by gypsyF or gypsyR paint is ∼5 Mb. (B) Representative images of CP190 IF and signals of gypsyF and gypsyR probes in dsRNA treated Kc cells. Cells were cultured at 25°C. Images are maximal projections of 26 Z-stacks. Nuclear edge is indicated with dashed line. Scale bar: 2 μm. (C) Probe volume relative to the fraction of nuclear volume. Left panel is control gypsyR probe, right is gypsyF probe. (D) Intermixing volume of gypsyF and gypsyR paints relative to nuclear volume. Data are from four biological replicates and a dot represents each replicate, n > 300 cells in each sample. Paired t-test was used, and error bars show standard deviation. ns, not significant; *P < 0.05 as indicated. (E) Nucleosome remodeller NURF301 cooperates with Su(Hw) to promote stable insulator complex binding at gypsy insulator sites to establish higher-order chromatin structure. After depletion of NURF301, Su(Hw) and CP190 cannot properly bind to gypsy binding sites, thus impairing gypsy insulator function and influencing chromatin organization.