QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 8 December 2005
doi: 10.1242/dev.02199

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, W. Articles by Johansen, K. M. Search for Related Content PubMed PubMed Citation Articles by Zhang, W. Articles by Johansen, K. M.

The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila

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

View larger version (26K): [in a new window]   Fig. 1. Histone H3K9 dimethylation spreads to ectopic locations in JIL-1 null and hypomorphic mutant backgrounds. (A-D) Polytene squash preparations labeled with anti-dmH3K9 antibody (red) and with Hoechst (DNA, blue). The upper panels show the anti-dmH3K9 labeling alone, whereas the lower panels show the composite images with the Hoechst labeling. The X chromosomes are indicated by X; the chromocenter with an arrow. In wild-type (wt) preparations, dmH3K9 labeling is mainly localized to the chromocenter (A). In JIL-1z2/JIL-1h9 (z2/h9, B) and in JIL-1z2 homozygous (z2/z2) male (C) and female (D) mutants, the dmH3K9 labeling is clearly upregulated on the X chromosomes and to a lesser extent on the autosomes. In the JIL-1 mutants the morphology of the polytene chromosomes is perturbed with coiled and compacted chromosome arms. (E) The level of histone H3S10 phosphorylation is greatly decreased in JIL-1 null and hypomorphic alleles, whereas the levels of dmH3K9, HP1, histone H3 and tubulin are comparable to wild type. Immunoblots were performed on extracts from wild-type (wt), JIL-1z2/JIL-1h9 (z2/h9) and JIL-1z2 homozygous (z2/z2) larvae. The immunoblots were labeled with anti-phospho-histone H3S10 (pH3S10), anti-dmH3K9, anti-HP1, anti-histone H3 (H3) and anti-tubulin antibodies.

View larger version (51K): [in a new window]   Fig. 2. HP1 and dmH3K9 are upregulated on both male and female X chromosomes in JIL-1 null mutants. (A-C) Confocal images of whole-mount polytene nuclei from male and female third instar larvae. The X chromosome is indicated by X; the chromocenter by an arrow. (A,B) Triple labeling with anti-HP1 (red) and anti-dmH3K9 (green) antibodies, and with Hoechst (DNA, blue), of female (A) and male (B) wild-type (wt) and JIL-1z2 homozygous (z2/z2) mutant polytene nuclei. (A) In the female, both HP1 and dmH3K9 are upregulated on the X chromosome, and to a lesser degree on the autosomes in JIL-1z2 homozygous nuclei. (B) In the male, both HP1 and dmH3K9 are upregulated on the X chromosome, and to a lesser degree on the autosomes in JIL-1z2 homozygous nuclei. (C) Triple labeling with anti-HP1 (red) and anti-MSL1 (green) antibodies, and with Hoechst (DNA, blue), of male wild-type (wt) and JIL-1z2 homozygous (z2/z2) mutant polytene nuclei. MSL-1 labeling identifies the male X chromosome. (D) Identification of the female X chromosome by a GFP-lacI fusion protein targeted to 256 lacO repeats inserted into the 4D5 interband region of polytene X chromosomes in a JIL-1z2 homozygous (z2/z2) mutant. The preparation was triple labeled with anti-dmH3K9 (red) and anti-GFP (green) antibodies, and with Hoechst (DNA, blue). The position of the lacO repeats identifying the X chromosome is indicated (arrowhead). Composite images (comp) are shown to the left.

View larger version (75K): [in a new window]   Fig. 3. The JIL-1Su(var)3-1[3] protein is mislocalized but does not affect dmH3K9 distribution or levels of histone H3S10 phosphorylation. (A,B) Triple labeling with anti-JIL-1 (red) and anti-dmH3K9 (A) or anti-MSL-1 (B) (green) antibodies, and with Hoechst (DNA, blue), of a male JIL-1z2/JIL-1Su(var)3-1[3] polytene squash preparation. The X chromosome is indicated by X; the chromocenter by an arrow. (A) The dmH3K9 antibody labeling is mainly localized to the chromocenter. (B) The JIL-1Su(var)3-1[3] protein is not upregulated on the X chromosome, which is identified by MSL-1 antibody labeling. (C) Higher magnification image of the preparation in B, showing the relative distribution of JIL-1 (red) and MSL-1 (green) antibody labeling. The JIL-1Su(var)3-1[3] protein is localized to the chromosomes, but the antibody labeling is diffuse and associates with both interband and banded regions. The arrows indicate regions on the X chromosome where the JIL-1Su(var)3-1[3] protein is localized to ectopic locations not overlapping with MSL-1 labeling. (D) The level of histone H3S10 phosphorylation in JIL-1z2/JIL-1Su(var)3-1[3] heteroallelic mutants is comparable to that in JIL-1z2/+ heteroalleles. Immunoblots were performed on extracts from wild-type (wt), JIL-1z2/+ (z2/+) and JIL-1z2/JIL-1Su(var)3-1[3] (z2/3-1) third instar larvae. The immunoblots were labeled with anti-phospho-histone H3S10 (pH3S10), anti-dmH3K9 and anti-histone H3 (H3) antibodies.

View larger version (65K): [in a new window]   Fig. 4. JIL-1 localization and kinase activity is not affected in Su(var)3-9 mutants. (A-D) Polytene squash preparations from male and female wild-type (wt) and Su(var)3-91/Su(var)3-92 (Su(var)3-9) third instar larvae labeled with JIL-1 antibody. Chromosome morphology and JIL-1 localization in Su(var)3-91/Su(var)3-92 preparations were similar to in wild-type preparations. (E) dmH3K9 levels were greatly reduced in Su(var)3-91/Su(var)3-92 mutants, whereas histone H3S10 phosphorylation was at wild-type levels. Immunoblots were performed on extracts from wild-type (wt) and Su(var)3-91/Su(var)3-92 (Su(var)3-9) third instar larvae. The immunoblots were labeled with anti-dmH3K9, anti-phospho-histone H3S10 (pH3S10) and anti-histone H3 antibodies. (F) dmH3K9 did not spread from the chromocenter to ectopic locations in polytene squashes from JIL-1z2/JIL-1z60 Su(var)3-91 (z2/z60, 3-9) larvae. The preparation was labeled with anti-dmH3K9 antibody (green) and Hoechst (DNA, blue). The X chromosome is indicated by X; the chromocenter by an arrow.