The epigenetic H3S10 phosphorylation mark is required for counteracting heterochromatic spreading and gene silencing in Drosophila melanogaster

Abstract

The JIL-1 kinase localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays with strong JIL-1 hypomorphic loss-of-function alleles have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components on position-effect variegation (PEV) and gene silencing. However, it is unclear whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein. By transgenically expressing various truncated versions of JIL-1, with or without kinase activity, and assessing their effect on PEV and heterochromatic spreading, we show that the gross perturbation of polytene chromosome morphology observed in JIL-1 null mutants is unrelated to gene silencing in PEV and is likely to occur as a result of faulty polytene chromosome alignment and/or organization, separate from epigenetic regulation of chromatin structure. Furthermore, the findings provide evidence that the epigenetic H3S10 phosphorylation mark itself is necessary for preventing the observed heterochromatic spreading independently of any structural contributions from the JIL-1 protein.

Fig. 1.

Expression of JIL-1 constructs transgenically in a wild-type background. (A) Diagrams of the JIL-1 CFP-tagged constructs analyzed. The region in the CTD where JIL-1^Su(var)3-1 alleles resulting in C-terminally truncated proteins have been mapped (Ebert et al., 2004) is indicated by a bracket. (B) Immunoblot labeled with JIL-1 antibody of protein extracts from wild-type (WT) and flies expressing the FL, the CTD and the ΔCTD constructs, respectively. Labeling with antibody against tubulin was used as a loading control. The relative migration of molecular size markers in kDa is indicated to the left of the immunoblot. (C) Immunoblot, labeled with antibody against phosphorylated H3S10 (H3S10ph) of protein extracts from salivary glands from WT third-instar larvae, from larvae expressing the FL, the CTD and the ΔCTD, respectively, and from JIL-1 null larvae (Z2). Labeling with antibody against histone H3 was used as a loading control.

Fig. 2.

The effect on PEV of the 118E-10 allele by expression of the CTD or the ΔCTD. (A) Examples of the degree of PEV in the eyes of wild-type JIL-1 flies (cont), wild-type JIL-1 flies expressing the CTD and wild-type JIL-1 flies expressing the ΔCTD in a 118E-10/118E10 background. All images are from male flies. (B) Histograms showing the levels of eye pigment of wild-type JIL-1 flies (cont), wild-type JIL-1 flies expressing the CTD and wild-type JIL-1 flies expressing the ΔCTD in a male 118E-10/118E10 background. The average pigment level when the CTD or the ΔCTD was expressed was compared with the control level using a two-tailed Student's t-test.

Fig. 3.

The effect on H3K9me2 localization in polytene chromosomes expressing the CTD. The polytene squash preparations were labeled with antibody against H3K9me2 (in red) and with Hoechst (DNA, in blue or grey). The X chromosome is indicated by an X. Preparations from wild-type (control) and male and female larvae expressing the CTD are shown. In wild-type preparations, H3K9me2 labeling was mainly localized to and abundant at the chromocenter – however, when the CTD was expressed, the H3K9me2 labeling spread to the autosomes and particularly to the X chromosome in both males and females.

Fig. 4.

The effect on PEV of the w^m4 allele by expression of the CTD or the ΔCTD. (A) Examples of the degree of PEV in the eyes of wild-type JIL-1 flies (cont), wild-type JIL-1 flies expressing the CTD and wild-type JIL-1 flies expressing the ΔCTD in a w^m4/Y background. (B) Histograms showing the levels of eye pigment of wild-type JIL-1 flies (cont), wild-type JIL-1 flies expressing the CTD and wild-type JIL-1 flies expressing the ΔCTD in a w^m4/Y background. The average pigment level when the CTD or the ΔCTD was expressed was compared with the control level using a two-tailed Student's t-test.

Fig. 5.

The effect on PEV of the 118E-10 allele in JIL-1 null flies expressing the FL, the CTD or the ΔCTD. (A) Examples of the degree of PEV in the eyes of JIL-1^z2/JIL-1^z2 null flies expressing the FL, the CTD or the ΔCTD in a 118E-10/+ background. All images are from male flies. (B) Histograms showing the levels of eye pigment of male JIL-1^z2/JIL-1^z2 null flies expressing the FL, the CTD or the ΔCTD in a 118E-10/+ background. The average pigment level when the CTD or the ΔCTD was expressed was compared with the level when the FL was expressed using a two-tailed Student's t-test.

Fig. 6.

Expression of transgenic JIL-1 constructs in JIL-1 null flies. (A) Immunoblot of protein extracts from wild-type (wt) and from JIL-1^z2/JIL-1^z2 null flies expressing the FL, the CTD and the ΔCTD, respectively (labeled with JIL-1 antibody). Labeling with tubulin antibody was used as a loading control. The relative migration of molecular size markers in kDa is indicated to the right of the immunoblot. (B) Immunoblot of protein extracts from salivary glands from wild-type third-instar larvae (wt) and from JIL-1^z2/JIL-1^z2 null larvae expressing the FL, the CTD and the ΔCTD, respectively [labeled with phosphorylated (H3S10ph) antibody]. Labeling with antibody against histone H3 was used as a loading control.

Fig. 7.

Chip analysis of the reporter gene hsp70-white in the 118E-10 P-element insertion. (A) Histograms of the relative enrichment of chromatin immunoprecipitated by antibody against phosphorylated H3S10 (H3S10ph) from the salivary glands of JIL-1^z2/JIL-1^z2 null third-instar larvae expressing the FL, the CTD or the ΔCTD in a 118E-10/+ background. For each experimental condition, the relative enrichment was normalized to the corresponding control immunoprecipitation with purified rabbit IgG antibody (cont). The graph shows the results from two independent experiments. (B) Histograms of the relative enrichment of chromatin immunoprecipitated by anti-H3K9me2 mAb from salivary glands of JIL-1^z2/JIL-1^z2 null third-instar larvae expressing the FL, the CTD or the ΔCTD in a 118E-10/+ background. For each experimental condition, the relative enrichment was normalized to the corresponding control immunoprecipitation with GST mAb 8C7 (cont). The graph shows the results from two independent experiments.

Fig. 8.

The effect on H3K9me2 localization in polytene chromosomes from JIL-1 null larvae expressing the FL, the CTD or the ΔCTD. The polytene squash preparations were labeled with antibody against H3K9me2 (in red) and with Hoechst (DNA, in blue or grey). The X chromosome is indicated by an X. Preparations from JIL-1^z2/JIL-1^z2 null larvae expressing either the FL, the ΔCTD or the CTD are shown. For comparison, the top panel shows a preparation from a JIL-1^z2/JIL-1^z2 null larvae without transgene expression.

Fig. 9.

The effect on H3K9me2 localization in polytene chromosomes from JIL-1 null larvae expressing a ‘kinase dead’ JIL-1 construct. The polytene squash preparation is from a male JIL-1 null (JIL-1^z2/JIL-1^z2) third-instar larvae triple labeled with Hoechst (DNA, in blue or grey), H3K9me2 antibody (in red) and JIL-1 antibody (in green). Note that although expression of the ‘kinase dead’ construct is near wild-type levels, and is localized on the chromosome arms and upregulated on the male X chromosome (X), the chromosome morphology as well as the spreading and upregulation of histone H3K9 dimethylation on the X chromosome are indistinguishable from those observed in JIL-1 null third-instar larvae (Fig. 7).