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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02960

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The chromodomain protein, Chromator, interacts with JIL-1 kinase and regulates the structure of Drosophila polytene chromosomes

Uttama Rath^*, Yun Ding^*, Huai Deng, Hongying Qi, Xiaomin Bao, Weiguo Zhang, Jack Girton, Jørgen Johansen and Kristen M. Johansen

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA

View larger version (61K): [in a new window]   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.

View larger version (34K): [in a new window]   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).

View larger version (38K): [in a new window]   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.

View larger version (33K): [in a new window]   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).

View larger version (101K): [in a new window]   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).

View larger version (64K): [in a new window]   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-1z2/JIL-1z2 (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.

View larger version (33K): [in a new window]   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.

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