Distribution of PAR and DAPI (A, C, and E) or PAR and ISWI (B, D, F, G, and H) on polytene chromosomes from wild-type (A, B, E, and F) and ISWI¹/ISWI² (C, D, G, and H) male third instar larvae. The different colours indicate DAPI (blue), ISWI (red), and PAR (green). (E and F) are magnifications of the boxed areas shown in (A and B), respectively. (G and H) are, respectively, magnifications of the autosome and X-chromosomal areas shown in (D). Arrows in (A and C) indicate X chromosomes.

(A) Larval nuclear extracts derived from the HA-6His-tagged ISWI line [6] were affinity purified on a His-Trap column [14] and 3% of the total input extract (I), supernatant (U), and 30% of the eluted fractions were subjected to Western blot analysis with aPAR (lanes 1–3) and aISWI (lanes 4–6) antibodies. The band pointed by the double arrow is lost in a mock purification.
(B) Upper panel: aPAR Western blot of purifed histones (lanes 1 and 2), recombinant p53 (lanes 3 and 4), ISWI (lanes 5 and 6), and NFκB p50 subunit (lanes 7 and 8) after incubation in presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of purified PARP. PARP was also incubated in the absence of any other potential substrate (lane 9). Lower panel: The same filter was stained with AuroDye (GE Healthcare) to reveal the blotted PARylated and non-PARylated proteins. The expected migrations of unmodified p50, p53, and ISWI are indicated by arrows.
(C) Upper panel: aPAR Western blot of 2 nmol each of full length ISWI (lanes 1 and 2), N-terminal truncated portion of ISWI (lanes 3 and 4; ISWI-N), C-terminal truncated portion of ISWI (lanes 5 and 6; ISWI-C) after incubation (15 min) in presence (lanes 2, 4, 6) or absence (lanes 1, 3, 5) of purified PARP (0.04 nmol). PARP was also incubated alone in the absence of substrate (lane 7). Lower panel: The same filter was stained with AuroDye (GE Healthcare) to reveal the blotted PARylated and non-PARylated proteins. The expected migrations of ISWI, ISWI-N, and ISWI-C are indicated by arrows, whereas PARP is indicated by asterisks.
(D) Larval nuclear extracts derived from larvae expressing TAP-tagged ISWI (ISWI-TAP) and control untagged extracts (ISWI) were affinity purified, as previously described [14]. The ISWI-CBP fusion, consisting of the ISWI protein fused in frame with the calmodulin binding peptide, was eluted from the resin by cleavage with the TEV protease. About 0.05% of the Input extract (I) and supernatant (U), 3% of the eluate (E) were subjected to Western blot analysis with aPAR and aISWI antibodies. Arrows indicate TAP-tagged ISWI (ISWI-TAP), ISWI fused in frame with the calmodulin binding peptide (ISWI-CBP), and endogenous untagged ISWI (ISWI).
(E) Immunoprecipitation with aPAR antibodies, of salivary gland extracts derived from wild type (lanes 1 and 3) and Parg^27.1 mutant (lanes 2 and 4) lines. Western blot analysis was performed on the immunoprecipitate with anti-PAR (lanes 1 and 2), anti-ISWI (lanes 3 and 4).
(F) Pull-down of FLAG-ISWI and purified PARP in the presence (lanes 5–8) or absence (lanes 1–4) of activating DNA. Proteins were detected by Western blot using aISWI and aPARP antibodies.
(G) Schematic representation of the domain structure of full length ISWI. The boundaries of the N-terminal (ISWI-N) and C- terminal (ISWI-C) truncated forms of ISWI are depicted.

Gel retardation assays of (A) mixed-length poly-nucleosomes purfied from chicken erythrocyte or in vitro assembled monoucleosomes (B) after incubation with increasing amounts of ISWI, in the presence or absence of PARP and in the presence or absence of PARP plus its competitive inhibitor 3-AB. For purified nucleosomes, 125 ng of a mixed length poly-nucleosome fraction were incubated with 2, 4, 8, and 16 nmol of ISWI, 0.4 nmol of PARP and 5 mM 3-AB. For reconstituted recombinant nucleosomes, 0.25 nmol of mononucleosomes were incubated with 0.5, 1, 2, and 4 nmol of ISWI, 0.2 nmol of PARP, and 2.5 mM 3-AB.
Activating DNA together with increasing amounts of ISWI were tested for the ability to shift (C) purified polynucleosomes and (D) recombinant mononucleosomes before and after incubation with PARP or PARP and 3-AB. Sixteen, and 32 nmol of ISWI, 0.4 nmol of PARP, and 40 mM 3-AB were used for purified nucleosomes, while 4 and 8 nmol of ISWI, 0.2 nmol of PARP, and 20 mM 3-AB were used for reconstituted recombinant nucleosomes. Numbers indicate mass ratio of ISWI over purified polynucleosomes (ISWI/Poly) or over in vitro assembled mononucleosomes (ISWI/Mono).

Distribution of PARP and DAPI (A, C, and E) or PARP and ISWI (B, D, F, G, and H) on polytene chromosomes from wild-type (A, B, C, and D) and ISWI¹/ISWI² (E, F, G, and H) male third instar larvae. PARP mainly localizes in the euchromatic interbands [30], while ISWI pattern contrasts with that of PARP and predominantly overlaps with DAPI stained bands, although a significant fraction of the protein is also associated with interbands [14]. The different colours indicate DAPI (blue), ISWI (red), and PARP (green). (C and D) are magnifications of the boxed areas shown in (A and B), respectively. (G and H) are, respectively, magnifications of the autosome and X-chromosomal areas shown in (F). Arrows in (A and E) indicate X chromosomes.

(A) Distribution of PAR (green) and ISWI (red) on polytene chromosomes from lines expressing UAS-GFP, UAS-ISWI, or UAS-ISWI^K159R transgenes under control of the eyGAL4 driver. Chromosomal DNA was visualized with DAPI (blue). All images were captured with the camera settings used for the UAS-GFP control. To measure directly the ISWI and PAR staining on polytene chromosomes, the levels of ISWI and PAR have been normalized with anti-Mod [14]. Our analysis revealed that on average there is an ∼8.6- and a ∼16.8-fold increase of PAR and ISWI staining, respectively, on polytene chromosomes overexpressing UAS-ISWI or UAS-ISWI^K159R, when compared to UAS-GFP chromosomes.
(B) Immunoprecipitation with anti-HA antibodies, of salivary gland extracts derived from lines expressing GFP, HA-tagged-ISWI, or -ISWI^K159R transgenes under control of eyGAL4. Western blot analysis was performed on the input (I: 3% of total), unbound (U: 3%), and pellet (P: 33%) fractions, with aPAR (lanes 1–9), aISWI (lanes 10–18), and aAlpha tubulin (as loading control) antibodies. Arrowhead indicates IgG.

(A) Dual assay for DNA-dependent ATP hydrolysis and poly-ADP-ribosylation (molar ratio ISWI/PARP ∼2.5). Black bars indicate percentage of total ATP hydrolysed, as measured by TLC analysis of an aliquot from each reaction mixture (1–6). Inclusion (+) or omission (-) of ISWI, DNA, PARP and 3-AB during the reaction is indicated. Aliquots of the same reaction mixtures were analysed by Western blot with the anti-PAR antibody. As a loading control, the same filter was stained with AuroDye. Results of analyses for the individual reaction mixtures are vertically aligned.
(B) Dual assay for chromatin-stimulated ATP hydrolysis and poly-ADP-ribosylation (molar ratio ISWI/PARP ∼2.5). Grey bars indicate % of total ATP hydrolysed, as measured by TLC analysis of an aliquot from each reaction mixture (1–6). Inclusion (+) or omission (-) of ISWI, chromatin (CHR), PARP, and 3-AB during the reaction is indicated. Aliquots of the same reaction mixtures were analysed by Western blot with anti-PAR antibody. As a loading control, the same filter was stained with AuroDye. Results of analyses for the individual reaction mixtures are vertically aligned.
(C) Time course of DNA-dependent ATP hydrolysis (molar ratio ISWI/PARP ∼133). Open diamonds (-): reactions contained PARP and ISWI, but not DNA. Black diamonds (D): reactions contained ISWI and DNA, but not PARP. Black triangles (D+P): reactions contained ISWI, PARP, and DNA. Open triangles (D+P+3-AB): reactions contained ISWI, PARP, DNA, and 3-AB. Open squares (D + 3-AB): reactions contained ISWI, DNA and 3-AB, but not PARP.
(D) Time course of chromatin-stimulated ATP hydrolysis (molar ratio ISWI/PARP ∼133). Open diamonds (-): reactions contained PARP and ISWI, but not chromatin. Black diamonds (C): reactions contained ISWI and chromatin, but not PARP. Black triangles (C+P): reactions contained ISWI, PARP and chromatin. Open triangles (C+P+3-AB): reactions contained ISWI, PARP, chromatin and 3-AB. Open squares (C + 3-AB): reactions contained ISWI, chromatin and 3-AB, but not PARP. The apparent mild ATPase inhibition exerted by PARP, under these conditions, is due to the very high ISWI/PARP molar ratio present in the reaction.

(A) Distribution of PAR (green) and ISWI (red) on polytene chromosomes from wild-type (wt) and homozygous Parp^C03256 (Parp) male third instar larvae. Chromosomes were also stained with DAPI to visualize DNA (blue). To measure directly the ISWI and PAR staining on polytene chromosomes, the levels of ISWI and PAR have been normalized with anti-Mod [14]. Our analysis revealed that on average there is a ∼4.1-fold decrease of the PAR staining in the Parp^C03256 mutant that is accompanied by a ∼6.6-fold increase in ISWI staining, when compared to wt chromosomes.
(B) ImmunoFISH using an aISWI antibody (green) and a DNA probe for the hsp70 loci at 87A and 87C (red) was carried out on polytene chromosomes prepared from wild-type third instar larvae maintained under non–heat-shocked conditions. Large panel shows the merged image for both signals. The image contains two examples of cytological loci 87A and 87C, which are indicated by arrows (large panel). Anti-ISWI immunostaining and in situ hybridization with fluorescently labeled probe are shown for magnifications of the regions harboring these loci (boxed areas), as both merged and split chromosome images (small panels). Arrowheads indicate the binding of ISWI at the 87A hsp70 locus.
(C) Co-immunolocalization of ISWI and PAR with DAPI staining on polytene chromosomes prepared from wild-type third instar larvae exposed to heat shock. Large panel shows the merged signals for ISWI (red) and PAR (green). A pair of heat shock puffs at 87A and 87C are indicated by arrows (large panel). Magnifications of the region containing both puffs are shown for DAPI, PAR, ISWI, and the ISWI/PAR merge (small panels). Arrowhead indicates loss of ISWI binding at the 87A hsp70 locus.
(D) Immunoprecipitation with aPAR antibody on wild-type native salivary gland extracts prepared under non–heat-shock (lanes 1, 2, 3, 7, 8, and 9) or heat-shock conditions (lanes 4, 5, 6, 10, 11, and 12). Western blot analysis was performed on the input (I: 3% of total), unbound (U: 3%), and pellet (P: 50%) fractions with anti-PAR (lanes 1–6), anti-ISWI (lanes 7–12), and anti-Tub (as loading control) antibodies. Arrowhead indicates IgG.