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-1^z2 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-1^z2 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-1^z2 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-1^z2 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-1^z2 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.

The JIL-1^{Su(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-1^z2/JIL-1^{Su(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-1^{Su(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-1^{Su(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-1^{Su(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-1^z2/JIL-1^{Su(var)3-1[3]} heteroallelic mutants is comparable to that in JIL-1^z2/+ heteroalleles. Immunoblots were performed on extracts from wild-type (wt), JIL-1^z2/+ (z2/+) and JIL-1^z2/JIL-1^{Su(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.