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First published online 16 January 2008
doi: 10.1242/dev.015362

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Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila

¹ Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
² Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA.

View larger version (6K): [in this window] [in a new window]   Fig. 1. Diagrams of the LacI-fusion constructs used for tethering to lacO repeat transgenic insertion lines. The JIL-1 histone H3S10 kinase is a 1207 amino acid protein with two kinase domains, KDI and KDII.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Ectopic tethering of lacI-JIL-1 fusion protein to a polytene band induces histone H3S10 phosphorylation and chromatin decondensation. (A-C) Triple labelings of polytene squash preparations from third instar Drosophila larvae homozygous for the lacO repeat line P11.3, which is inserted into the middle of a polytene band in region 96C1-2. GFP-LacI was tethered to the lacO repeats in A and LacI-JIL-1 in B,C. GFP-, LacI- and JIL-1-antibody labeling is shown in green, H3S10ph-antibody labeling in red and Hoechst 33258 labeling of DNA in blue or gray. The white arrows indicate the lacO repeat insertion site. The polytene chromosomes from the three preparations are aligned to show the `split' in the polytene bands, reflecting decondensation of the chromatin when lacI-JIL-1 fusion protein is tethered to the band, in contrast to its wild-type morphology, when GFP-LacI is tethered and there is no ectopic upregulation of histone H3S10 phosphorylation. Note: the endogenous JIL-1 and H3S10ph antibody labeling is too weak relative to the LacI-JIL-1 signal and the induced hyperphosphorylation of H3S10 to be clearly visible at this exposure level. (D) Without a GAL4-driver line there is no LacI expression or changes to the band/interband structure. Double labelings with LacI antibody (in green) and Hoechst 33258 (in blue or gray) of polytene squash preparations from third instar larvae homozygous for the lacO repeat line P11.3 and containing a LacI-JIL-1 transgene but without a GAL-4 driver. Arrows indicate the approximate lacO repeat insertion sites.

View larger version (65K): [in this window] [in a new window]   Fig. 3. Tethering of LacI-JIL-1 to heterozygous lacO repeat insertion lines. (A-C) Polytene squash preparations double labeled with JIL-1 antibody (green) and either Hoechst 33258 or propidium iodide (red or gray). Heterozygous preparations for the band insertion line P11.3 (A), the band-interband insertion line P19.9 (B) and the interband insertion line 4D5 (C) are shown. (D) Heterozygous preparation for the interband insertion line 4D5 triple labeled with LacI antibody in green, H3S10ph antibody in red and with Hoechst 33258 labeling of DNA in blue or gray. Arrowheads in B,C point to bands of compacted chromatin that are decondensed on the polytene chromosome half where LacI-JIL-1 was targeted. A tub-GAL4 driver was used in A-D.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Tethering of LacI-JIL-1 `kinase dead' to lacO repeat insertion lines. (A) Double labeling of a polytene squash preparation homozygous for the lacO repeat line P11.3 with LacI antibody (in green) and with Hoechst 33258 (in blue and gray). The arrow indicates chromatin structure perturbations at the insersion site. Arrowheads indicate chromatin structure perturbations and ectopic contacts at other sites. (B) Triple labeling of a polytene squash preparation homozygous for the lacO repeat line P11.3 with LacI antibody (in green), with H3S10ph antibody (in red) and with Hoechst 33258 (in blue and gray). The upper panel shows LacI-JIL-1 tethering compared to LacI-JIL-1 kinase dead tethering in the lower panel. Arrows indicate the approximate lacO repeat insertion sites. (C) Triple labeling of a polytene squash preparation heterozygous for the lacO repeat lines P11.3 and 4D5 with LacI antibody (in green), with JIL-1 antibody (in red) and with Hoechst 33258 (in blue and gray). Arrows indicate the approximate lacO repeat insertion sites. (D) Levels of H3S10 phosphorylation were reduced when LacI-JIL-1 kinase dead was expressed, compared with wild-type levels and when LacI-JIL-1 was expressed in homozygous lacO P11.3 lines. Immunoblots were performed on extracts from third instar larval salivary glands and labeled with JIL-1, H3S10ph, histone H3 and tubulin antibodies. The upper arrow indicates LacI-JIL-1 or LacI-JIL-1-kd, whereas the lower arrow shows the location of wild-type JIL-1.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Tethering of LacI-JIL-1 to lacO repeat insertion lines using a late-onset Sgs3-GAL4 driver. The figure shows polytene squash preparations double labeled with LacI antibody (green) and propidium iodide (red or gray). (A) Homozygous preparation for the band insertion line P11.3. (B) Heterozygous preparation for the interband insertion line 4D5. Arrows in A,B point to areas of compacted chromatin that were decondensed where LacI-JIL-1 was targeted.

View larger version (77K): [in this window] [in a new window]   Fig. 6. Tethering of LacI-JIL-1 is not associated with upregulation of either Pol II0ser2, Pol II0ser5, histone H4K16 acetylation or of the BRM complex at the LacO insertion site. Triple labelings with JIL-1 antibody (in green), Pol II0ser2 antibody (A) or Pol II0ser5 antibody (B) (in red), and Hoechst 33258 (in blue or gray) of polytene squash preparations from larvae homozygous for the lacO repeat line P11.3. (C) Triple labeling with JIL-1 antibody (in green), histone H4K16ac antibody (in red) and Hoechst (in blue or gray) of a polytene squash preparation from a male third instar larvae homozygous for the lacO repeat line P19.9. The upregulation of histone H4K16 acetylation on the male X chromosome (X) is clearly evident in comparison to the normal autosomal level at the tethering site (arrows). (D) Triple labelings with LacI antibody (in green), Brahma antibody (in red) and Hoechst 33258 (in blue or gray) of polytene squash preparations from larvae homozygous for the lacO repeat line P11.3.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Model for the establishment of a euchromatic chromatin state by ectopic H3S10 phosphorylation. (A) lacO repeats (in red) inserted into a polytene band region with condensed chromatin in the absence of LacI-JIL-1 expression. The region has normal band-interband morphology. (B) When LacI-JIL-1 is expressed, the extended region of lacO repeats recruits high levels of LacI-JIL-1, which in turn hyperphosphorylates histone H3S10 at the target site as well as at adjacent chromatin regions. The ectopic phosphorylation of histone H3S10 subsequently induces the release of condensing factors and/or recruits chromatin remodeling factors, resulting in a euchromatic chromatin state at and near the insertion site.

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