Chromator and JIL-1 kinase directly interact

The interphase localization of Chromator to polytene chromosome interband regions (Rath et al., 2004; Eggert et al., 2004; Gortchakov et al., 2005) is very similar to that reported for the JIL-1 histone H3S10 kinase (Jin et al., 1999; Jin et al., 2000; Wang et al., 2001). For this reason we explored whether the two proteins were present in the same macromolecular complex by performing co-immunolocalization and biochemical studies. Fig. 1 shows confocal images of a double labeling with JIL-1- and Chromator-specific antibodies of a third-instar larval polytene chromosome squash preparation. JIL-1 localization is shown in green, Chromator protein localization in red, and co-localization is indicated by yellow regions in the composite image. The predominantly yellow labeling in the composite image indicates that JIL-1 colocalizes with Chromator extensively along the entire length of the chromosome arms (Fig. 1A). At some band locations in the composite image the color is more green or orange than pure yellow as a result of slight differences in the relative staining intensity of the two antibodies, which can vary from preparation to preparation. However, in all cases examined the banding patterns themselves appeared identical (Fig. 1B,C) providing evidence that Chromator and JIL-1 are co-localized at most if not all interband regions.

In order to probe further for a potential interaction we performed immunoprecipitation (IP) experiments using S2 cell lysates. For these experiments, proteins were extracted from S2 cells, immunoprecipitated using either JIL-1- or Chromator-specific antibodies, fractionated on SDS-PAGE after the IP, immunoblotted, and probed with antibodies to Chromator and JIL-1, respectively. Fig. 2A shows an IP experiment using an anti-Chromator antibody where the immunoprecipitate is detected by JIL-1 antibody as a 160 kDa band that is also present in the S2 cell lysate. This band was not present in the lane where only immunobeads were used for the IP (Fig. 2A). Fig. 2B shows the converse experiment: JIL-1 antiserum immunoprecipitated a 130 kDa band detected by Chromator antibody that was also present in S2 cell lysate but not in control IPs with immunobeads only. Furthermore, we stably and transiently transfected S2 cells with a V5-tagged fulllength Chromator contruct and prepared nuclear extracts from lysed cells. Fig. 2C shows an IP experiment using V5 antibody where a 160 kDa band detected by the anti-JIL-1 antibody that was also present in the S2 cell nuclear extract was co-immunoprecipitated. This band was not present in the lane where the V5 antibody IP was performed in nuclear extracts from untransfected control S2 cells (Fig. 2C). These results strongly indicate that Chromator and the JIL-1 kinase are present in the same protein complex.

To characterize further the interaction between Chromator and JIL-1 and to identify the domains mediating the interaction we performed overlay assays with GST-fusions of various regions of the two proteins. For the initial screening of JIL-1 we used four GST-fusion proteins covering the N-terminal domain (JIL-1-NTD), the first kinase domain (JIL-1-KDI), the second kinase domain (JIL-1-KDII), and the C-terminal domain (JIL-1-CTD) as illustrated in Fig. 3A. For Chromator we generated an N-terminal construct including the chromodomain (Chro-NTD) as well as a C-terminal construct (Chro-CTD) (Fig. 3A). Fig. 3B,C shows JIL-1 GST-fusion proteins that were coupled with glutathione agarose beads, washed, fractionated by SDS-PAGE, transferred to nitrocellulose paper, and incubated with glutathione-agarosebead-purified Chro-CTD and Chro-NTD GST-fusion constructs, respectively. Protein interactions were detected with Chromator C-terminal mAb 6H11 in Fig. 3B and with the N-terminal Chromator mAb 12H9 in Fig. 3C. As illustrated in Fig. 3B (arrows) only the JIL-1-CTD and Chro-CTD fusion constructs were found to interact in these assays. Western blot analysis of the GST proteins purified in these experiments and detected with anti-GST antibody showed that similar levels of JIL-1-NTD, JIL-1-KDI, JIL-1-KDII and JIL-1-CTD fusion proteins were present in these assays (Fig. 3D). Immunoblots of the purified Chro-CTD and Chro-NTD fusion constructs used for the overlays are shown in Fig. 3B (lane 6) and Fig. 3C (lane 7), respectively. The region of JIL-1 that was found to interact with Chromator, the JIL-1 CTD-domain, can be further divided into two distinct regions (Bao et al., 2005): an acidic region from residues 887-1033 that has a predicted pI <4 and a basic region from residues 1034-1207 that has a pI >11 (Fig. 3A). Thus, in order to define the sequences of JIL-1 responsible for the molecular interaction between JIL-1 and Chromator, we generated GST fusion proteins comprising these two regions, JIL-1-CTD-A and JIL-1-CTD-B (Fig. 3A), and performed overlay experiments with the Chro-CTD construct as described above. As shown in Fig. 3E the JIL-1-CTD and JIL-1-CTD-A fusion proteins both interacted with Chro-CTD as detected by Chromator mAb 6H11 (arrows) whereas JIL-1-CTD-B or GST alone did not. Western blot analysis of the GST proteins purified in these experiments and detected with GST-antibody showed that approximately equivalent levels of JIL-1-CTD, JIL-1-CTD-A, and JIL-1-CTD-B fusion proteins were present in these assays (Fig. 3F). An immunoblot of the purified Chro-CTD fusion construct used for the overlay is shown in Fig. 3E (lane 5). Taken together these results suggest that the interaction between JIL-1 and Chromator is direct and that it is mediated by sequences in the C-terminal domain of Chromator and by the acidic region within the C-terminal domain of JIL-1.

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Fig. 1.

Co-localization of JIL-1 with Chromator at polytene chromosome bands. Double labeling of a female polytene chromosome squash preparation with antibodies against JIL-1 (B) and Chromator (C). The composite image (A) shows the extensive overlap between JIL-1 (green) and Chromator (red) labeling at a large number of chromosome bands as indicated by the predominantly yellow color. The images are from confocal sections.

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Fig. 2.

Chromator and JIL-1 immunoprecipitation assays. (A) Immunoprecipitations (ip) of lysates from S2 cells were performed using Chromator antibody (mAb 6H11, lane 4) and JIL-1 antibody (Hope antiserum, lane 3) coupled to immunobeads or with immunobeads only as a control (lane 2). The immunoprecipitations were analyzed by SDS-PAGE and western blotting using JIL-1 pAb for detection. JIL-1 antibody staining of S2 cell lysate is shown in lane 1. JIL-1 is detected in the JIL-1 and Chromator immunoprecipitation samples as a 160 kDa band (lane 3 and 4, respectively) but not in the control sample (lane 2). (B) Immunoprecipitations of lysates from S2 cells were performed using Chromator antibody (mAb 6H11, lane 3) and JIL-1 antibody (Hope antiserum, lane 4) coupled to immunobeads or with immunobeads only as a control (lane 2). The immunoprecipitations were analyzed by SDS-PAGE and western blotting using Chromator mAb 6H11 for detection. Chromator antibody staining of S2 cell lysate is shown in lane 1. Chromator is detected in the JIL-1 and Chromator immunoprecipitation samples as a 130 kDa band (lane 4 and 3, respectively) but not in the control sample (lane 2). The relative migration of molecular size markers are indicated in kDa. (C) Immunoprecipitations of nuclear extracts from S2 cells were performed using V5 antibody from cells transfected with a V5-tagged full-length Chromator (lane 2) or from untransfected cells as a control (lane 3). The immunoprecipitations were analyzed by SDS-PAGE and western blotting using JIL-1 antiserum for detection. JIL-1 antibody staining of S2 cell nuclear extract is shown in lane 1. JIL-I is detected as a 160 kDa band in V5-antibody immunoprecipitates from V5-tagged Chromator transfected S2 cells (lane 2) but not in the untransfected control samples (lane 3).

Generation of hypomorphic Chromator alleles

We have previously demonstrated that the P-element insertion KG03258 is a lethal loss-of-function mutation in the Chro gene (Rath et al., 2004). Unfortunately, most homozygous KG03258 animals die as embryos and none survive past the first-instar larval stages, thus precluding the analysis of polytene chromosome structure in third-instar larval salivary gland cells. Attempts to generate new hypomorphic loss-of-function Chro alleles that survive to third-instar larval stages by imprecise P-element excisions have so far been unsuccessful (Rath et al., 2004; Gortchakov et al., 2005). In addition, such studies are likely to be complicated by the close proximity of the essential neighboring ssl1 gene to the Chro locus (Rath et al., 2004; Gortchakov et al., 2005). For these reasons we generated EMS-induced point mutations in the Chro gene using standard protocols (Grigliatti, 1986). We identified a total of 12 new alleles that reduced adult survival rates by more than 50% when heterozygous with the KG03258 allele compared with a wild-type allele. Complementation tests between the newly generated alleles revealed that individuals heteroallelic for two of these alleles survive to third-instar larval stages. These two alleles were subsequently sequenced and further characterized in this study. The Chro⁷¹ allele is comprised of a G to A nucleotide change at nucleotide position 402 of the Chro coding sequence that introduces a premature stop codon resulting in a truncated 71 amino acid protein (Fig. 4A). The truncated N-terminal fragment does not contain the chromodomain and Chro⁷¹ probably acts as a strong hypomorphic or null allele. Chro⁷¹ is homozygous embryonic lethal with no first-instar larval escapers. The Chro⁶¹² allele consists of a C to T nucleotide change at nucleotide position 2024 that introduces a premature stop codon resulting in a truncated 612 amino acid protein that retains the chromodomain (Fig. 4A). Chro⁶¹² is homozygous embryonic lethal with a few first-instar larval escapers. However, Chro⁷¹/Chro⁶¹² transheterozygotes survived to third-instar larval stages although no larvae have been observed to pupate. This suggests that Chro⁶¹² is a severe hypomorphic loss-of-function allele that nonetheless in combination with Chro⁷¹ can provide partial function sufficient for development to third-instar stages. Although genetic crosses were performed to replace the other chromosomes it should be noted that the presence of second site mutations on the third chromosome cannot be ruled out and may account for the early lethality of homozygous Chro⁶¹² mutants. However, the effect of such potential mutations are probably masked in the Chro⁷¹/Chro⁶¹² transheterozygotes. The immunoblot of protein extracts from wild-type and Chro⁷¹/Chro⁶¹² third-instar larvae in Fig. 4B demonstrates that no detectable full-length Chromator protein was present in the mutant larvae. The immunoblot was labeled with Chromator-specific mAb 6H11 (Rath et al., 2004) which was generated to C-terminal sequence deleted in both the Chro⁷¹ and Chro⁶¹² alleles.

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Fig. 3.

Mapping of the JIL-1 interaction domain with Chromator. (A) Diagrams of the JIL-1 and Chromator proteins indicating the domains to which GST-fusion proteins were made for mapping. In the overlay experiments various truncated JIL-1 GST-fusion protein constructs to the domains in A or a GST-only control were fractionated by SDS-PAGE, western blotted, incubated with Chro-CTD (B) or Chro-NTD (C) GST-fusion protein, and interactions detected with the C-terminal Chromator mAb 6H11 (B) or the N-terminal Chromator mAb 12H9 (C). The only interaction detected was between the JIL-1-CTD and Chro-CTD fusion proteins (arrows in B). Immunoblots of the overlay GST-fusion proteins Chro-CTD and Chro-NTD are shown (B, lane 7 and C, lane 6, respectively). (D) Immunoblot of the input GST-fusion proteins used for the overlay experiments in B and C detected with the anti-GST mAb 8C7. (E) Overlay experiments with truncated C-terminal JIL-1 GST-fusion protein constructs to the subdomains shown in A or a GST-only control were fractionated by SDS-PAGE, western blotted, incubated with Chro-CTD, and interactions detected with the C-terminal Chromator mAb 6H11. In these experiments interactions between Chro-CTD and JIL-1-CTD as well as JIL-1-CTD-A were detected (arrows) but not between Chro-CTD and JIL-1-CTD-B. An immunoblot of the overlay GST-fusion protein Chro-CTD is shown in lane 5. (F) Immunoblot of the input GST-fusion proteins used for the overlay experiments in E detected with the anti-GST mAb 8C7. This defined the JIL-1 C-terminal acidic domain as sufficient for mediating interactions with the C-terminal domain of Chromator. The relative migration of molecular size markers is indicated to the right of the immunoblots in kDa.

Polytene chromosome structure is disrupted in hypomorphic Chromator mutants

The generation of severely hypomorphic Chro alleles that as transheterozygotes survived to third-instar larval stages allowed the analysis of their effect on polytene chromosome structure. Fig. 4C,D shows a comparison of polytene squashes from wild-type and Chro⁷¹/Chro⁶¹² larvae labeled with Hoechst 33258 stain. Whereas wild-type polytene chromosomes show extended arms with a regular pattern of Hoechst-stained bands (Fig. 4C), this pattern is severely perturbed in Chro⁷¹/Chro⁶¹² mutant larvae (Fig. 4D). In the latter preparations band/interband regions were disrupted and the chromosome arms were coiled and condensed (Fig. 4D). To understand the underlying causes of these defects we performed an ultrastructural analysis by preparing squashes of polytene chromosomes from Chro⁷¹/Chro⁶¹² third-instar larvae for transmission electron microscopy (TEM) and comparing them with squashes from wild-type larvae (Fig. 5). Fig. 5A shows the orderly segregation into interband and the more electron-dense banded regions in TEM of a wild-type autosome. However, in Chro⁷¹/Chro⁶¹² mutants the alignment of the chromatids in the interbands was disrupted and the orderly arrangement of compacted chromatin in the banded regions was severely affected as well (Fig. 5B,C). Another feature of the phenotype was the folding and coiling of the chromosomes with numerous ectopic contacts connecting non-homologous regions (Fig. 5C). However, despite these disruptions, distinct band and interband regions were still clearly discernable in the mutant chromosomes. These findings suggest that normal Chromator function is required for maintaining the orderly segregation of bands and interbands in polytene chromosome structure.

Localization of Chromator and JIL-1 in mutant polytene chromosomes

The finding that Chromator and JIL-1 directly interact within the same protein complex raised the question whether one of the proteins is required for the polytene chromosome localization of the other. To address this issue we performed double labelings of polytene squashes from wild-type, Chro⁷¹/Chro⁶¹², and JIL-1^z2/JIL-1^z2 third-instar larvae with the JIL-1 pAb Hope and the Chromator mAb 6H11. The JIL-1^z2 allele is a true null that when homozygous produces no JIL-1 protein (Wang et al., 2001; Zhang et al., 2003). Banded regions of the polytene chromosomes were labeled with Hoechst 33258. Fig. 6A (upper panel) shows that in wild-type polytene squashes both Chromator and JIL-1 colocalize to interband regions in a pattern complementary to the Hoechst labeling. In Chro⁷¹/Chro⁶¹² mutants the polytene chromosome morphology is perturbed; however, JIL-1 continued to be localized to interband regions not labeled by Hoechst (Fig. 6A, middle panel). No full-length Chromator protein was detected by mAb 6H11, which recognizes a C-terminal epitope (Rath et al., 2004) in these preparations. In the JIL-1 null mutants with no detectable JIL-1 protein the chromosome morphology was also severely perturbed as previously described (Wang et al., 2001; Deng et al., 2005); nonetheless, Chromator still localized to chromosome regions in a complementary pattern to the Hoechst labeling of compacted chromatin (Fig. 6A, bottom panel). These data strongly suggest that Chromator localization to the polytene chromosomes does not depend on the JIL-1 protein. However, JIL-1 dependence on the Chromator protein could not be ruled out by these experiments because the Chro⁶¹² allele can give rise to a truncated Chromator protein that contains the chromodomain and that has enough C-terminal sequence to potentially recruit JIL-1. The Chro⁷¹ allele is unlikely to play such a role because it lacks the entire C-terminal domain and because the N-terminal region of Chromator is without a functional nuclear localization signal (Rath et al., 2004). Labelings of Chro⁷¹/Chro⁶¹² mutant polytene squashes with the N-terminal Chromator mAb 12H9 showed that Chro⁶¹² protein indeed localizes to the interband regions of the polytene chromosomes (Fig. 6B). We therefore used RNAi methods in S2 cells to completely deplete Chromator levels and assayed for the consequences on JIL-1 localization by labeling with JIL-1 antibody. In control cells, as shown in Fig. 7 JIL-1 antibody specifically labels the chromatin and is upregulated on the X chromosome (the S2 cell line is a `male' cell line) as previously described (Jin et al., 1999; Jin et al., 2000; Wang et al., 2001). To verify that it was indeed the X chromosome that had upregulated JIL-1 levels the preparations were double labeled with anti-MSL-1 antibody. MSL-1 is a member of the MSL (male-specific lethal) dosage compensation complex and is found only on the male X chromosome (Kuroda et al., 1991; Kelley and Kuroda, 1995). However, the distribution of JIL-1 did not change in Chro RNAi-treated cell cultures (Fig. 7A, bottom panel) where Chromator protein levels were reduced below limits detectable by immunoblot analysis (Fig. 7B). These results suggest that Chromator is not required for JIL-1 localization to chromatin nor for upregulation of JIL-1 on the male X chromosome. Similar results were also obtained for male Chro<sup>71</sup>/Chro<sup>612</sup> mutant polytene chromosomes labeled with JIL-1 antibody (Fig. 6C). Furthermore, although the phenotype of JIL-1 and Chro<sup>71</sup>/Chro<sup>612</sup> mutant polytene chromosomes generally resemble each other, a notable difference is that the morphology of the male X chromosome in Chro<sup>71</sup>/Chro<sup>612</sup> larvae was similar to that of the autosomes (Fig. 6C) and not `puffed' as in JIL-1 null mutants (Wang et al., 2001; Deng et al., 2005). Although these experiments indicate that Chromator does not directly recruit JIL-1 to polytene chromosomes the presence of truncated Chromator proteins in the Chro⁷¹/Chro⁶¹² mutant does not allow us to completely exclude the possibility of an indirect requirement.

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Fig. 4.

EMS induced Chro alleles. (A) Diagram of the wild-type Chromator protein and the potential truncated protein products of the EMS induced Chro alleles, Chro⁷¹ and Chro⁶¹². The Chro⁷¹ allele is comprised of a G to A nucleotide change at nucleotide position 402 of the Chro coding sequence that introduces a premature stop codon resulting in a truncated 71 amino acid protein. The Chro⁶¹² allele consists of a C to T nucleotide change at nucleotide position 2024 that introduces a premature stop codon resulting in a truncated 612 amino acid protein that retains the chromodomain. (B) Chromator protein expression in Chro⁷¹/Chro⁶¹² mutant third-instar larvae compared with wild type larvae. The immunoblots were labeled with the C-terminal Chromator mAb 6H11 and with anti-tubulin antibody as a loading control. Full-length Chromator is detected as a 130 kDa protein by mAb 6H11 in wild-type larvae; however, no full-length Chromator is detectable in the mutant larvae. The relative migration of molecular weight markers is indicated to the left of the immunoblots in kDa. (C,D) Polytene chromosome preparations from third-instar larvae were labeled with Hoechst to visualize the chromatin. Preparations are shown from a wild-type female larvae (C) and from a female Chro⁷¹/Chro⁶¹² mutant larvae (D). Reduced levels of wild-type Chromator protein have a severe effect on the structure and organization of larval polytene chromosomes. Note the disruption and misalignment of interband and banded regions and the extensive coiling and folding of the chromosome arms in Chro⁷¹/Chro⁶¹² mutant chromosomes (D).

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Fig. 5.

Ultrastructure of Chro⁷¹/Chro⁶¹² mutant polytene chromosomes. (A) TEM micrograph of a wild-type polytene chromosome. Note the clear segregation into bands and interbands and the orderly alignment of euchromatic chromatid fibrils. (B,C) Chromosomes from Chro⁷¹/Chro⁶¹² polytene salivary gland nuclei. The micrograph in B shows the disorganization and misalignment of band/interband polytene chromosome regions (arrows). The micrograph in C shows the folding and coiling of the chromosomes with numerous ectopic contacts connecting non-homologous regions (arrows).

Genetic interactions between Chro and JIL-1 alleles

To determine whether Chromator and JIL-1 genetically interact in vivo we explored interactions between mutant alleles of Chro and JIL-1 by generating double-mutant individuals. Since Chro and JIL-1 both are located on the third chromosome we first recombined each of the Chro⁷¹ and Chro⁶¹² alleles onto the JIL-1^z2 chromosome. Subsequently, JIL-1^z2 Chro⁷¹/TM6 Sb Tb males were crossed with JIL-1^z2 Chro⁶¹²/TM6 Sb Tb virgin females generating JIL-1^z2 Chro⁷¹/JIL-1^z2 Chro⁶¹² progeny. In control experiments we crossed JIL-1^z2/TM6 Sb Tb males with JIL-1^z2/TM6 Sb Tb virgin females generating JIL-1^z2/JIL-1^z2 progeny as well as Chro⁷¹/TM6 Sb Tb males with Chro⁶¹²/TM6 Sb Tb virgin females generating Chro⁷¹/Chro⁶¹² progeny. In these crosses the TM6 chromosome was identified by the Tb marker. Consequently, the experimental genotypes could be distinguished from balanced heterozygotic larvae by being non-Tb and the expected mendelian ratio of non-Tb to Tb larvae would be 1:2 because TM6/TM6 is embryonic lethal. Table 1 shows that individuals of both the Chro⁷¹/Chro⁶¹² and JIL-1^z2/JIL-1^z2 genotype develop into third-instar larvae in numbers that were not statistically different (P>0.9, χ²-test) from the expected mendelian ratio with that of TM6 balanced heterozygotes. However, in the JIL-1^z2 Chro⁷¹/JIL-1^z2 Chro⁶¹² double-mutant combination there was a clear statistically significant difference (P<0.001, χ²-test) as no non-Tb third-instar larvae were observed. This suggests that a simultaneous reduction in both JIL-1 and Chromator function synergistically reduces viability during development and is consistent with the hypothesis that the two proteins interact in vivo.

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Fig. 6.

Localization of JIL-1 and Chromator in mutant polytene chromosomes. (A) Triple labelings with the JIL-1 pAb Hope (green), the C-terminal Chromator mAb 6H11 (red) and Hoechst 33258 (blue) of polytene squashes from wild-type (upper panel), Chro⁷¹/Chro⁶¹² (middle panel), and JIL-1^z2/JIL-1^z2 (lower panel) female third-instar larvae. The composite image (comp) is shown to the left. The mAb 6H11 epitope is not present in either of the truncated Chro⁷¹ or Chro⁶¹² proteins. (B) Double labeling with the N-terminal Chromator mAb 12H9 (red) and Hoechst 33258 (blue) of polytene squashes from a Chro⁷¹/Chro⁶¹² female third-instar larvae. The composite image (comp) is shown to the left. (C) Labeling with the JIL-1 pAb Hope of polytene squashes from wild-type (left micrograph) and Chro⁷¹/Chro⁶¹² (right micrograph) male third-instar larvae. Note the upregulation of JIL-1 on the male X chromosome (X) of both wild-type and Chro⁷¹/Chro⁶¹² mutant larvae.

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Fig. 7.

RNAi depletion of Chromator in S2 cells does not affect JIL-1 chromosome localization. (A) Triple labelings with the JIL-1 pAb Hope (green), anti-MSL-1 antibody (red) and Hoechst 33258 (blue) of Chromator dsRNA treated S2 cells (lower panel) and mock-treated control cells (upper panel). The composite image (comp) is shown to the left and the location of the X chromosome is indicated with an X. (B) Western blot with Chromator mAb 6H11 of control-treated and Chromator RNAi-treated S2 cells from the cultures shown in A. In the RNAi sample Chromator protein levels (Chro) was substantially reduced compared with the level observed in the control cells. Tubulin levels (tub) are shown as a loading control.

Drosophila stocks and generation of new Chro alleles

Fly stocks were maintained according to standard protocols (Roberts, 1998). Canton-S was used for wild-type preparations. The lethal Chro P-element insertion allele KG03258 is described in Rath et al. (Rath et al., 2004) and the JIL-1^z2 null allele is described in Wang et al. (Wang et al., 2001) and in Zhang et al. (Zhang et al., 2003). New Chro mutant alleles were generated by ethyl methyl sulfonate (EMS) mutagenesis using standard procedures (Grigliatti, 1986). In this screen w¹¹¹⁸ males that had wild-type alleles of Chro were treated with 25 mM EMS in a 1% sucrose solution for 18-20 hours. EMS-treated males were then mass mated with w¹¹¹⁸ TM2 Ubx e/TM6 Sb Tb e virgin females. Male w¹¹¹⁸/Y;^*/TM6 Sb Tb e progeny from these mass matings were selected and individually mated with y w¹¹¹⁸ KG03258/TM6 Sb Tb e virgin females. The progeny of these single male matings were screened for potential new Chro alleles that reduced adult survival rates by more than 50% when heterozygous with the KG03258 allele. Twelve such new Chro alleles were identified in this screen and outcrossed for six generations to w¹¹¹⁸ TM2 Ubx e/TM6 Sb Tb e flies to eliminate non-specific second-site mutations on the other chromosomes. Complementation tests between the newly generated alleles revealed that individuals heteroallelic for two of these alleles, Chro⁷¹ and Chro⁶¹², survive to third-instar larval stages. The molecular lesions of Chro⁷¹ and Chro⁶¹² were determined by PCR mapping and sequencing as described (Zhang et al., 2003). Recombinant JIL-1^z2 Chro⁷¹ and JIL-1^z2 Chro⁶¹² chromosomes were generated by standard crosses as described (Ji et al., 2005) and the genotypes confirmed by PCR analysis (Zhang et al., 2003). Balancer chromosomes and markers are described (Lindsley and Zimm, 1992).

Antibodies

The Chromator-specific mAbs 6H11 and 12H9 have been previously characterized (Rath et al., 2004) and the anti-MSL-1 rabbit antiserum was the generous gift of M. Kuroda (Harvard Medical School, Boston, MA) and R. Kelley (Baylor College of Medicine, Houston, TX). The affinity-purified Hope rabbit anti-JIL-1 polyclonal antibody was described in Jin et al. (Jin et al., 1999) and the anti-GST mAb 8C7 in Rath et al. (Rath et al., 2004). The anti-α-tubulin and anti-V5 antibodies were obtained from commercial sources (Sigma-Aldrich and Invitrogen, respectively).

Immunohistochemistry and ultrastructural analysis

Polytene chromosome squash preparations were performed as in Kelley et al. (Kelley et al., 1999) using the 5 minute fixation protocol and labeled with antibody as described (Jin et al., 1999). S2 cells were affixed onto poly-L-lysine coated coverslips and fixed with Bouin's fluid for 10 minutes at 24°C and methanol for 5 minutes at -20°C. The cells on the coverslips were permeabilized with PBS containing 0.5% Triton X-100 and incubated with diluted primary antibody in PBS containing 0.1% Triton X-100, 0.1% sodium azide and 1% normal goat serum for 1.5 hour. Double and triple labelings using epifluorescence were performed using various combinations of antibodies and Hoechst 33258 to visualize the DNA. The appropriate species- and isotype-specific Texas Red-, TRITC- and FITC-conjugated secondary antibodies (Cappel/ICN, Southern Biotech) were used (1:200 dilution) to visualize primary antibody labeling. The final preparations were mounted in 90% glycerol containing 0.5% n-propyl gallate. The preparations were examined using epifluorescence optics on a Zeiss Axioskop microscope and images were captured and digitized using a high-resolution Spot CCD camera. Confocal microscopy was performed with a Leica confocal TCS NT microscope system equipped with separate Argon-UV, Argon, and Krypton lasers and the appropriate filter sets for Hoechst, FITC, Texas Red and TRITC imaging. A separate series of confocal images for each fluorophor of double-labeled preparations were obtained simultaneously with z-intervals of typically 0.5 μm using a PL APO 100×/1.40-0.70 oil objective. Images were imported into Photoshop where they were pseudocoloured, image processed and merged. In some images non-linear adjustments were made for optimal visualization of Hoechst labeling of chromosomes. For ultrastructural studies we prepared polytene chromosome squash preparations of wild-type and Chro⁷¹/Chro⁶¹² third-instar larvae according to the procedure of Semeshin et al. (Semeshin et al., 2004) as described in Deng et al. (Deng et al., 2005).

SDS-PAGE and immunoblotting

SDS-PAGE was performed according to standard procedures (Laemmli, 1970). Electroblot transfer was performed as described (Towbin et al., 1979) with transfer buffer containing 20% methanol and in most cases including 0.04% SDS. For these experiments we used the Bio-Rad Mini PROTEAN II system, electroblotting to 0.2 μm nitrocellulose and using anti-mouse HRP-conjugated secondary antibody (Bio-Rad) (1:3000) for visualization of primary antibody diluted 1:1000 in Blotto. The signal was visualized using chemiluminescent detection methods (SuperSignal kit, Pierce). The immunoblots were digitized using a flatbed scanner (Epson Expression 1680). Immunoblot analysis of Chro⁷¹/Chro⁶¹² mutants was performed as described (Wang et al., 2001; Zhang et al., 2003) using extracts from third-instar larvae with wild-type larvae as controls.

Overlay experiments

The four truncated GST-JIL-1 fusion proteins, JIL-1-NTD (residues 1-211), JIL-1-KDI (residues 251-554), JIL-1-KDII (residues 615-917) and JIL-1-CTD (residues 927-1207) have been previously described (Jin et al., 2000) and the constructs JIL-1-CTD-A (residues 887-1033) and JIL-1-CTD-B (residues 1034-1207) were described in Bao et al. (Bao et al., 2005). Two Chromator GST-fusion proteins, Chro-NTD (residues 1-346) and Chro-CTD (residues 329-926) were cloned into the pGEX4T vector using standard techniques (Sambrook and Russell, 2001). The respective GST-fusion proteins were expressed in XL1-Blue cells (Stratagene) and purified over a glutathione agarose column (Sigma-Aldrich) according to the pGEX manufacturer's instructions (Amersham Pharmacia Biotech). For the overlay interaction assays approximately 2 μg GST or of the appropriate JIL-1 GST-fusion proteins were fractionated by SDS-PAGE and electroblotted to nitrocellulose. The blots were subsequently incubated with approximately 2 μg of either the Chro-NTD or the Chro-CTD GST-fusion protein overnight at 4°C in PBS with 0.5% Tween-20 on a rotating wheel. The blots were washed four times for 10 minutes each in PBS with 0.5% Tween-20 and binding detected by antibody labeling with either Chromator mAb 6H11 or 12H9. Input proteins were analyzed by SDS-PAGE and immunoblotting with GST-antibody.

Immunoprecipitation assays

For co-immunoprecipitation experiments, anti-JIL-1 or anti-Chromator antibodies were coupled to protein-A beads (Sigma) as follows: 10 μl of affinity-purified Hope anti-JIL-1 serum or 10 μl mAb 6H11 was coupled to 30 μl protein-A-Sepharose beads (Sigma) for 2.5 hours at 4°C on a rotating wheel in 50 μl IP buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.1% Triton X-100, 0.1% Nonidet P-40, 1 mM Phenylmethylsulfonyl fluoride and 1.5 μg aprotinin). The appropriate antibody-coupled beads or beads only were incubated overnight at 4°C with 200 μl S2 cell lysate on a rotating wheel. Beads were washed three times for 10 minutes each with 1 ml IP buffer with low-speed pelleting of beads between washes. The resulting bead-bound immunocomplexes were analyzed by SDS-PAGE and western blotting according to standard techniques (Harlow and Lane, 1988) using mAb 6H11 to detect Chromator and Hope antiserum to detect JIL-1. For V5-antibody immunoprecipitation experiments in S2 cells we used a full-length Chromator (926 aa) construct with an in-frame V5 tag at the C-terminal end previously described by Rath et al. (Rath et al., 2004). The S2 cells were transfected with this construct using a calcium phosphate transfection kit (Invitrogen) and expression was induced by 0.5 mM CuSO₄. Cells expressing the Chromator construct or mock-transfected control cells were harvested 18-24 hours after induction. Nuclear extracts were prepared as described (Smith et al., 2000), immunoprecipitated with 10 μl anti-V5 antibody coupled to 30 μl protein-A-Sepharose beads as described above, fractionated by SDS-PAGE, and immunoblotted using Hope JIL-1 antiserum for detection.

RNAi interference

dsRNAi in S2 cells was performed according to Clemens et al. (Clemens et al., 2000) and as described in Rath et al. (Rath et al., 2004). A 780 bp fragment encoding the 5′ end of Chromator cDNA was PCR amplified and used as templates for in vitro transcription using the Megascript™ RNAi kit (Ambion). 40 μg of synthesized dsRNA was added to 1×10⁶ cells in six-well cell culture plates. Control dsRNAi experiments were performed identically except pBluescript vector sequence (800 bp) was used as a template. The dsRNA-treated S2 cells were incubated for 6-7 days and then processed for immunostaining and immunoblotting. For immunoblotting, 10⁵ cells were harvested, resuspended in 50 μl S2 cell lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl and 1% Nonidet P-40), boiled and analyzed by SDS-PAGE and western blotting with anti-Chromator antibody (mAb 6H11) and anti-α-tubulin antibody.