1. Poly(ADP-ribosyl)ation Regulates Insulator Function and Intrachromosomal Interactions in Drosophila 
Chin-Tong Ong, Kevin Van Bortle, Edward Ramos, Victor G. Corces 
Cell 
Volume 155, Issue 1, Pages (September 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Drosophila Insulators Undergo PARylation In Vivo and In Vitro
(A–C) CP190, Su(Hw), and Mod(mdg4)2.2 are PARylated in S2 cells. Cell lysates (Lys) were immunoprecipitated with either preimmune serum (IgG) or antibodies that recognize different insulator proteins. The various fractions were subjected to western analysis with PAR antibody followed by antibodies to different insulator proteins. Black arrowheads point to the possible PARylated form of Mod(mdg4)2.2 protein.
(D) CP190 and dCTCF can be PARylated in vitro. GST, GST-tagged CP190, and GST-tagged dCTCF were PARylated in vitro using biotin-NAD+ as a substrate in the presence or absence of 3AB. In vitro products were western blotted with Streptavidin-HRP, followed by dCTCF and CP190 antibodies.
(E) The K566 residue within the putative PBZ domain is essential for PARylation of CP190. GST, GST-tagged CP190, and GST-tagged CP190:K566A proteins were PARylated in vitro and western blotted with streptavidin-HRP and CP190 antibody. Asterisk indicates the location of CP190 in the gel. The sequence of the PBZ domain and the location of the K566A mutation are indicated at the bottom of the panel.
(F) CP190:K566A protein is not PARylated in vivo. Lysates from S2 cells transfected with WT or K566A mutant (KA) CP190-myc constructs were immunoprecipitated with myc antibody and probed with PAR and CP190 antibodies.
See also Figure S1.
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4. Figure 2
Mutation of the Parp Gene Affects gypsy and Fab-8 Insulator Activity
(A) The level of abdomen pigmentation is inversely correlated to insulator activity at the y2 locus. Percentage of flies with different levels of pigmentation in y2; +/+, y2; ParpCH1/+, y2; CP1904-1/CP190H312 and y2; CP1904-1/CP190H312-ParpCH1 lines. Flies were examined for y2 expression 1 day after eclosion. Chi-square test: p < (+/+ and ParpCH1/+) and p = 0.04 (ParpCH1/+ and CP1904-1/CP190H312-ParpCH1).
(B) The severity of the cut wing margin phenotype correlates with insulator activity at the ct6 locus. Percentage of flies with different levels of cut wing margin phenotypes in ct6; +/+, ct6; ParpCH1/+, ct6; mod(mdg4)T6 and ct6; mod(mdg4)T6/mod(mdg4)T6-ParpCH1 lines. Chi-square test: p < between mod(mdg4)T6 and mod(mdg4)T6/mod(mdg4)T6-ParpCH1.
(C) Eyes of male flies of the genotype Fab /+; +/+, Fab /+; CP190H312/+, Fab /+; ParpCH1/+, Fab /+; CTCFy+6/+ and Fab /+; CP190H312ParpCH1/+. Eye color was examined 1 hr after eclosion.
(D) Amount of red eye pigment extracted from the eyes of male flies of the respective genotypes. Blue bars indicate the presence of the Fab /+ transgene. Mean absorbance at OD 485 nm and SD. At least 23 animals from each genotype were assayed.
(E) WT, but not K566A transgene, restores insulator activity at the ct6 locus in null CP190H312/P11 flies. Top: the majority of the flies from transgenic strain Wt258 have a cut wing, whereas most transgenic KA122 flies have wing margins that resemble those of hypomorphic CP1904-1/H312 (hypo) flies. Chi-square test: p < Bottom: western blot of lysate from five adult flies of different genotypes.
See also Figure S1.
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5. Figure 3
Interaction between Insulator Proteins and Their Association with the Nuclear Lamina Are Stabilized by PARylation
(A) PARylation stabilizes interactions between CP190 and dCTCF proteins. Lysate (Lys) from control (Ct) and 3AB-treated cells was immunoprecipitated with preimmune serum (IgG) or CP190 antibody (CP190). These fractions were subjected to western blot analysis with CP190 and dCTCF antibodies. Quantification of the relative level of dCTCF protein that was pulled down by CP190 antibody and SD from six independent experiments (∗∗p < 0.005).
(B) CP190:K566A protein interacts weakly with dCTCF in S2 cells. Lysates from cells transfected with WT or KA construct were immunoprecipitated with myc antibody and probed with CP190 and dCTCF antibodies. Quantification of the relative level of dCTCF protein that was pulled down by myc antibody and SD from three independent experiments (∗∗p < 0.008).
(C) PARylation promotes the association of insulator proteins with the nuclear lamina. Nucleus, nuclear matrix, and soluble fractions isolated from control and 3AB-treated cells were western blotted with CP190, Su(Hw), Mod(mdg4)2.2, Lamin Dm0, and histone H3 antibodies. Mean band intensities quantified by ImageJ software and SD from at least five independent experiments (∗∗∗CP190, p = , n = 6; Su(Hw), p = , n = 7; Mod(mdg4)2.2, p = , n = 5).
(D) Formation of CP190 insulator bodies is impaired in Parp03256 mutant larvae. Immunolocalization of CP190 (green) and Lamin Dm0 (red) in diploid nuclei from imaginal wing disc cells with DNA stained by DAPI (blue). Histogram depicting the distribution of the nuclear CP190 staining pattern in OR (WT) and Parp03256 mutant larvae.
See also Figure S2.
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6. Figure 4
A Subset of Insulator Binding Sites Is Regulated by PARylation
(A) Comparison of CP190 ChIP-seq peaks across an 840 kb region of Drosophila chromosome 3R in control (Ct) and 3AB-treated S2 cells. Arrow indicates site at which CP190 binding is disrupted by 3AB treatment.
(B) Graphs representing the number of Su(Hw), CP190, dCTCF, and Mod(mdg4)2.2 binding sites that exhibit greater than 2-fold changes between Ct and 3AB samples (left). Right: Venn diagram of the overlap between different 3AB-downregulated insulator sites. The asterisk represents a site where binding of four insulator proteins was reduced by 3AB treatment.
(C) Distribution of genomic CP190 binding sites (green) and sites with more than 3-fold reduction in the 3AB treated cells (blue) with respect to TSS. Each interval on the x axis represents a 200 bp window.
(D) Graph representing the distance between adjacent genomic (green), 3AB-downregulated (blue), and randomly picked (black) CP190 sites.
(E) Percentage of independent and aligned insulator binding sites affected by 3AB treatment. Genome-wide refers to insulator binding sites that contain consensus motifs and are bound by different insulator proteins. Ct > 3AB refers to 3AB-downregulated insulator binding sites.
(F) Percentage of genome-wide and 3AB-downregulated insulator sites that are within 2 kb of TAD borders.
See also Figure S3.
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7. Figure 5
Intrachromosomal Interactions between Specific Distant CP190 Binding Sites Are Regulated by PARylation
(A) Bait A represents an aligned insulator site with dCTCF and Su(Hw) consensus motifs. Left: ChIP signal of four insulator proteins in control cells (top). Relative ChIP-qPCR of CP190 and dCTCF at the bait and the SD from four independent experiments (bottom). Right: graphical depiction of the intrachromosomal interactions between bait A and 12 CP190 binding sites in control cells. 3AB-responsive interactions are represented by a green line. Middle: ChIP signal of CP190 surrounding bait A in control (CT) and 3AB-treated cells. Graph of relative crosslinking frequency between bait A and the four affected sites in control and 3AB samples and SD from four independent experiments.
(B) Bait B represents an independent dCTCF site that is bound by CP190 protein, ChIP-qPCR validation, and SD from four independent experiments (left). 19 intrachromosomal interactions between the bait B fragment and other CP190 binding sites were validated in control cells (middle); 6 of these interactions were reduced by 3AB treatment with error bars indicating the SD from 4 independent experiments (right).
(C) Bait C represents a class of CP190 binding sites devoid of DNA consensus motifs for BEAF-32, Su(Hw), and dCTCF proteins. Left: validation of CP190 binding at bait C by ChIP-qPCR with SD from seven independent experiments. 18 intrachromosomal interactions between bait C and other CP190 binding sites were validated in control cells (middle) of which two interactions were downregulated by 3AB. Error bars indicate SD from four independent experiments (right).
∗p < 0.05, ∗∗p < 0.02, and ∗∗∗p <
See also Figures S4 and S5.
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8. Figure 6
Model of How PARylation Regulates Insulator-Mediated Chromosome Organization
PARylation at CP190 K566 promotes its interaction with dCTCF. PARylation modulates the binding of insulator proteins within TADs, which in turn affects the intrachromosomal interactions between distant insulator sites and their association with the nuclear lamina.
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9. Figure S1
Drosophila Insulators Undergo In Vivo and In Vitro PARylation, Related to Figures 1 and 2
(A) dCTCF is PARylated in S2 cells. Left: Lysates (Lys) were immunoprecipitated with either preimmune serum (IgG) or antibody that recognizes dCTCF protein. The various fractions were Western-blotted with PAR antibody (10H) followed by dCTCF antibody. Right: Lysates were immunoprecipitated with mouse IgG or antibody that recognizes the PAR moiety on proteins (10H) and western-blotted with dCTCF antibody.
(B) Mod(mdg4)2.2 and Su(Hw) can be PARylated in vitro. GST, GST-tagged Mod(mdg4)2.2 and Su(Hw) were PARylated in vitro using NAD+ as a substrate in the presence or absence of 3-aminobenzamide (3AB). In vitro products were Western-blotted with 10H and GST antibodies.
(C) Alignment of CP190 and dCTCF with proteins characterized by the presence of the PBZ motif (blue). ∗: The score was calculated with the BLOSUM62 scoring matrix (Stothard, 2000) using human APLF-1 as the reference sequence. Consensus: [K/R]xxCx[F/Y]GxxCxbbxxxxHxxx[F/Y]xH where b denotes a basic residue.
(D) Expression of wild-type and K566A mutant CP190-6xMyc constructs in S2 cells. Left: Schematic of the expression vector and the experiment. Right: Western blot analyses of lysate isolated from untransfected and transfected cells.
(E) Percentage of flies with different level of abdominal pigmentation in y2; mod(mdg4)T6,e and y2; mod(mdg4)T6,e/mod(mdg4)T6,e-ParpCH1 flies. Flies were examined 1 day after eclosion. Chi-square test: p <
(F) Mutations in the Parp gene affect Fab-8 insulator activity. Top: Crosses used to generate F1 male offspring for Fab-8 insulator assays. Bottom: Eyes of male flies of the genotypes Fab /+, Fab /CP190H312, Fab /ParpCH1, Fab / CTCFy+6 and Fab /CP190H312ParpCH1 (top row), Fab /+; +/+, Fab /+; CP190H312/+, Fab /+; ParpCH1/+, Fab /+; CTCFy+6/+ and Fab /+; CP190H312ParpCH1/+ (bottom row). Eye color was examined 1 hr after eclosion.
(G) Overview of the P-element vector containing wild-type and K566A CP190 transgene and the mating scheme used to generate transgenic lines for analyses.
(H) CP190 transgene carrying the K566A mutation cannot rescue the insulator activity at ct6 locus in null CP190H312/P11 flies. Top: Phenotype of ct6 wings in different genetic backgrounds. The level of insulator activity is reflected through the number of notches (red arrow head) at the wing margin. Bottom: Percentage of transgenic flies with different wing phenotypes. Chi-square test between wild-type and K566A transgenic lines is p < (left). Western analyses of the level of CP190 and Lamin Dm0 proteins in different transgenic lines (right).
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10. Figure S2
Inhibition of PARylation by 3AB Treatment and Parp Knockdown Affects Insulator Protein-Protein Interaction and Their Localization to the Nuclear Lamina, Related to Figure 3
(A) Immunolocalization of Parp (green) and CP190 (red) in polytene chromosomes from wild-type Oregon R (OR) larvae. DNA is stained with DAPI (blue).
(B) Morphology of S2 cells 16 hr after control or 3AB treatment.
(C) Effective inhibition of PARylation in S2 cells by 3AB. Lysate from control (Ct) and 3AB treated cells was immunoprecipitated with anti-PAR antibody (10H) and subjected to Western blot analysis with PAR antibody.
(D) 3AB treatment inhibits PARylation of CP190. Lysates (Lys) from control and 3AB treated cells were immunoprecipitated with CP190 antibody. The different fractions were Western blotted with PAR followed by CP190 antibodies.
(E) Western-blotting of lysates (Lys) isolated from control (Ct) and 3AB treated cells with antibodies against CP190, dCTCF and Mod(mdg4)2.2.
(F) Immunolocalization of CP190 (green) and Mod(mdg4)2.2 (red) in polytene chromosomes from OR larvae (top) and Parp03256 mutant larvae (bottom). DNA is stained with DAPI (blue).
(G) Association of CP190 and dCTCF proteins to the nuclear lamina is partially affected in the absence of PARylation. Nuclear, matrix, and soluble fractions isolated from control (Ct) and 3AB treated cells were Western blotted with CP190, dCTCF, Lamin Dm0 and histone H3 antibodies. Mean band intensities quantified with ImageJ software and standard deviation from two independent experiments (∗p < 0.03).
(H) Effective knockdown (KD) of the Parp gene in S2 cells by dsRNA treatment. Lysates from β-galactosidase (βgal) and Parp knockdown cells were western-blotted with PARP (left), PAR (right) and Lamin Dm0 antibodies (bottom).
(I) Interaction between CP190 and dCTCF is stabilized by PARylation. Lysates (Lys) from β-galactosidase (βgal) and Parp knockdown cells were immunoprecipitated with CP190 antibody. These fractions were probed with CP190 and dCTCF antibodies. Quantification of the relative level of dCTCF protein that was pulled-down by CP190 antibody and standard deviation from two independent experiments (∗p < 0.05).
(J) PARylation promotes the association of insulator proteins with the nuclear matrix. Whole nuclear and nuclear matrix fractions isolated from β-galactosidase (βgal) and Parp knockdown cells were western-blotted with CP190, Su(Hw), dCTCF, Mod(mdg4)2.2 and Lamin Dm0 antibodies. Mean band intensities quantified with ImageJ software and standard deviation from two independent experiments (∗p < 0.05; ∗∗p < 0.005).
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11. Figure S3
Inhibition of PARylation Only Affects a Subset of Insulator Binding Sites, Related to Figure 4
(A) Comparison of the raw reads of dCTCF (top) and Mod(mdg4)2.2 (bottom) ChIP-seq on chromosome 3R in control (Ct) and 3-aminobenzamide (3AB) treated S2 cells.
(B) Pie chart representing the fraction of invariant and 3AB-responsive CP190, dCTCF, Su(Hw) and Mod(mdg4)2.2 binding sites. Insulator peaks in control and 3AB samples were assigned with MACS and analyzed with computational tools available in Galaxy.
(C) MEME logos representing the binding motif of genomic (top) and 3AB-responsive (bottom) insulator sites. Left: dCTCF and right: Su(Hw).
(D) Examples of qPCR validation of sites that exhibited significant reduction in CP190 binding in 3AB-treated cells. Each panel compares the ChIP-seq peak assigned by MACS (arrow, left), the raw reads from the wiggle file (arrow, right) and qPCR result (bottom). Control (Ct) is blue and 3AB treated sample is red. Mean ChIP-qPCR values and standard deviation (SD) from at least three independent experiments (∗∗p < 0.005).
(E) qPCR quantification of myc-ChIP at selected CP190 binding sites in S2 cells transfected with either wild-type (WT) or K566A mutant CP190 construct. Mean ChIP-qPCR values and standard deviation (SD) from two independent experiments (∗∗∗p = 0.002; ∗∗p = 0.006; ∗p = 0.02).
(F) Distribution of genome-wide (green) and 3AB-responsive (blue) dCTCF and Mod(mdg4)2.2 insulator binding sites with respect to transcription start sites (TSSs). Each interval on the x axis represents a 200-bp window.
(G) Graph representing the distance between adjacent dCTCF and CP190 sites. Green line between adjacent genomic sites; blue line between adjacent 3AB-downregulated sites and black line between adjacent randomly-picked sites.
(H) Percentage of CP190 sites associated with different histone modifications as determined by k-means clustering of the indicated histone marks found ± 2 kb from the summit of the unaffected and 3AB-downregulated CP190 binding sites.
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12. Figure S4
ChIP-Seq Profiles of Four Insulator Proteins across the Three Bait Fragments Used in 4C Experiments, Related to Figure 5
Comparison of the raw reads from wiggle files of CP190, Mod(mdg4)2.2, dCTCF and Su(Hw) ChIP-seq across the bait fragments in control (Ct) and 3AB treated samples. Left: Bait A is an aligned insulator site with DNA consensus motifs for dCTCF and Su(Hw). Binding of four insulator proteins is reduced by 3AB treatment. Middle: Bait B is an independent dCTCF site where binding of CP190 and dCTCF is reduced in 3AB treated cells. Right: Bait C is a CP190 binding site that lacks DNA consensus motifs for BEAF-32, Su(Hw) and dCTCF. Consequently, only relatively weak Su(Hw) and dCTCF signal can be detected across the bait fragment.
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13. Figure S5 Overview of 4C Experiments, Related to Figure 5
(A) Scheme of the 4C experiment. The specificity of primer pairs is confirmed by the presence of amplified DNA in the 3C sample but not in the non-ligated control (NL) after nested inverse PCR.
(B) Agarose gels stained with ethidium bromide showing the first inverse PCR and nested PCR products of bait fragments A and B.
(C) DNA amplified after 30 cycles of nested PCR reactions using different dilutions of the first inverse PCR product as a template. There is no product in non-ligated control (NL). Gel quantification of nested products show that the amplification occurs within the linear range at 1/80 dilution (equivalent to 0.3 ng).
(D) An example of confirmation of 3C interactions using a site-specific primer with the anchor primer on the bait. Approximately 0.2 ng of the first inverse PCR product were used as the template for each validation. “NL” refers to the amplified DpnII cut, non-ligated DNA; “4C” refers to the amplified 3C DNA and “4C+DpnII” refers to the amplified 3C DNA that was re-digested with DpnII. A 3C interaction is validated by the presence of high PCR yield in “4C”, no product in “NL” and weak PCR intensity in the “4C+DpnII” lane.
(E) An example illustrating the identification of 3C interactions affected by 3AB treatment. Five independent 3C libraries were generated from control (Ct) and 3AB-treated (3AB) S2 cells (Expt1-5). Approximately 0.2 ng of the first inverse PCR product from each library were then used for amplification. The effect of 3AB was confirmed when at least four out of the five libraries showed similar pattern in the PCR yield. Top: 3AB-affected interaction. Middle: 3C interaction unaffected. Bottom: PCR with reference primers that quantified copy numbers of ligated products. qPCR was then used to tabulate the graph in Figure 5.
(F) Relative cross-linking frequency of sites C1 and C2 with bait fragment C in the β-galactosidase (βgal) and CP190 knockdown S2 cells. Mean 4C-qPCR values and standard deviations (SD) from 3 independent cross-linked samples are shown.
(G) Intra-chromosomal interactions between bait 28 and surrounding CP190 binding sites are not downregulated by 3AB. Top left: ChIP signal of four insulator proteins at bait 28. Top right: Mean ChIP-qPCR values of CP190 (n = 8) and dCTCF (∗p < 0.05, n = 6) at bait 28 in control and 3AB treated cells with standard deviations (SD). Bottom: Graphical depiction of the intra-chromosomal interactions between bait 28 and 16 CP190 binding sites and the CP190 ChIP signal across the homeotic bithorax complex locus (n = 3).
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