H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

Abstract

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w^m4 PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.

Introduction

In the absence of H3S10 phosphorylation by the JIL-1 kinase the major heterochromatin markers H3K9me2, HP1a and Su(var)3–7 spread to ectopic locations on the chromosome arms of Drosophila polytene chromosomes.^1-3 These observations suggested a model for a dynamic balance between euchromatin and heterochromatin,^1,3-5 where the level of gene expression is determined by antagonistic functions of the euchromatic H3S10ph mark and the heterochromatic H3K9me2 mark.^3,5-8 Wang et al.⁵ tested this model by transgenically expressing various truncated versions of JIL-1, with or without kinase activity, and correlating their effect on PEV with the levels of the H3S10ph and H3K9me2 marks at a hsp70-white reporter gene as determined by ChIP assays in the pericentric insertion line 118E-10. At pericentric sites loss-of-function alleles of JIL-1 act as enhancers of PEV whereas gain-of-function alleles act as suppressors.⁷ As predicted by the model, the results of Wang et al.⁵ showed that the level of the H3K9me2 mark at the reporter was inversely proportional to the H3S10ph level. PEV was enhanced with increased levels of H3K9me2 in the absence of H3S10 phosphorylation and PEV was suppressed with increased levels of the H3S10ph mark and a concomitant decrease in the level of the H3K9me2 mark.⁵ However, it has been demonstrated that JIL-1 can act both as an enhancer as well as a suppressor of w^m4 PEV depending on the precise levels of JIL-1,^3,6 and that the genetic interactions between JIL-1 and the Su(var)3–9 and Su(var)2–5 alleles in regulating PEV of w^m4 are more complex than in the case of 118E-10 where reduced levels of JIL-1 always act as an enhancer.^7,8Therefore, in the present study we explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis.

Results and Discussion

The In(1)w^m4 X chromosome contains an inversion that juxtaposes the euchromatic white gene and centric heterochromatic sequences distal to the nucleolus organizer (Fig. 1).^9,10 The resulting somatic variegation of w^m4 expression occurs in clonal patches in the eye reflecting heterochromatic spreading from the inversion breakpoint that silences w^m4 expression in the white patches and euchromatic packaging of the w gene in those patches that appear red (reviewed in ref. 11) (Fig. 2B). Studies of this effect suggest that the degree of spreading may depend on the amount of heterochromatic factors at the breakpoint (reviewed in refs. 12 and 13). Interestingly, strong hypomorphic combinations of JIL-1 alleles, in which heterochromatic factors spread to ectopic locations,^1,2 act as suppressors not enhancers of PEV of the w^m4 allele (Fig. 2E).⁶ Based on these findings, Lerach et al.⁶ proposed a model where the suppression of PEV of w^m4 in strong JIL-1 hypomorphic backgrounds is due to a reduction in the level of heterochromatic factors at the pericentromeric heterochromatin near the inversion breakpoint site that reduces its potential for heterochromatic spreading and silencing (Fig. 1).

Figure 1. Diagrams of the distribution of heterochromatic factors relative to the white gene in wild-type and w^m4 X chromosomes. (A) In wild-type high levels of heterochromatic factors are confined to pericentric heterochromatin (in black) and the distally located white gene is expressed at normal levels. (B) In w^m4 an inversion juxtaposes the white gene to the pericentric heterochromatin where spreading of heterochromatic factors across the inversion breakpoint (in dark gray) represses white expression as reflected in PEV. (C) In a JIL-1 null w^m4 background in the absence of H3S10 phosphorylation there is a redistribution of heterochromatic factors from the pericentric heterochomatin to ectopic locations on the chromosome arms (light gray). The resulting decrease in the level of heterochromatic factors at the pericentric heterochromatin (in dark gray) reduces its potential for heterochromatic spreading and gene silencing and under these conditions the white gene is expressed.

Figure 2. The effect on PEV of the w^m4 allele by JIL-1 hypomorphic alleles or by expression of the CTD or the ΔCTD. Examples of the degree of PEV in the eyes of (A) wild-type flies, (B) w^m4 flies, (C) w^m4 flies expressing the ΔCTD, (D) w^m4; JIL-1^z2/+ flies, (E) w^m4; JIL-1^z2/ JIL-1^z60 flies and (F) w^m4 flies expressing the CTD. All pictures are from male flies. PEV of the w^m4 allele in the strong hypomorphic JIL-1^z2/ JIL-1^z60 background is shown in (E) because JIL-1^z2/ JIL-1^z2 null mutant backgrounds have no adult escapers.

In order to measure how the relative H3S10ph and H3K9me2 levels at the white gene were affected in the experiments, we performed ChIP assays as in Legube et al.¹⁴ and in Wang et al.⁵ Chromatin was immunoprecipitated (ip) from larval salivary glands from wild-type, the inversion line w^m4, the inversion line in either a heterozygous JIL-1 mutant background w^m4; JIL1^z2/+ or a homozygous JIL-1 mutant background w^m4; JIL-1^z2/JIL-1^z2, or the inversion line with either a transgenic JIL-1 CTD or ΔCTD construct w^m4; “JIL-1 transgene”/+; da-GAL4/+ using rabbit anti-H3S10ph antibody or purified rabbit IgG antibody (negative control) or mAbs to H3K9me2 or GST (negative control). Primers that amplify region -110 to +65 of the white gene were used to amplify the precipitated material. Experiments were done in triplicate and relative enrichment of white DNA from the H3S10ph and H3K9me2 ips were normalized to the corresponding control antibody ips performed in tandem for each experimental sample. Statistical comparisons using the various genotypes are provided in Tables 1 and 2. As illustrated in Figure 3 there is a relatively higher enrichment of H3S10ph (4.3 ± 0.8) than of H3K9me2 (2.0 ± 0.5) at the white gene in wild-type. However, compared with wild-type (Fig. 2A) the variegated eye phenotype in w^m4 (Fig. 2B) is correlated with an almost 4-fold decrease in relative H3S10ph enrichment levels (1.4 ± 0.4) and a more than 3-fold increase in the relative H3K9me2 enrichment levels (6.7 ± 1.6). Interestingly, this distribution can be reversed by expression of a JIL-1 ΔCTD construct which lacks the COOH-terminal sequences required for proper chromatin localization leading to mislocalization of the protein.¹⁵ However, it does retain its kinase activity resulting in ectopic histone H3S10 phosphorylation¹⁵ and suppression of PEV (Fig. 2C).⁵ As shown in Figure 3 expression of JIL-1 ΔCTD in a w^m4 background leads to a dramatic increase of almost 10-fold in the relative enrichment of H3S10ph (9.9 ± 0.9) at the white gene accompanied with a decrease in the relative enrichment of H3K9me2 (1.7 ± 0.1) to levels comparable to wild-type (Tables 1 and 2).

Table 1. Statistical comparison of H3K9me2 levels.

Genotype	wm4	wm4; z2/+	wm4; z2/z2	wm4; CTD	wm4; ΔCTD
wt	p < 0.001	p < 0.001	p > 0.4	p > 0.5	p > 0.8
wm4	-	p > 0.9	p < 0.001	p < 0.001	p < 0.001
wm4; z2/+	-	-	p < 0.001	p < 0.001	p < 0.001
wm4; z2/z2	-	-	-	p > 0.8	p > 0.5
wm4; CTD	-	-	-	-	p > 0.7

For each genotype the average relative enrichment from three sets of measurements were compared using a two-tailed Student’s t-test.

Table 2. Statistical comparison of H3S10ph levels.

Genotype	wm4	wm4; z2/+	wm4; z2/z2	wm4; CTD	wm4; ΔCTD
wt	p < 0.005	p < 0.002	p < 0.002	p < 0.001	p < 0.0001
wm4	-	p > 0.9	p > 0.7	p > 0.5	p < 0.0001
wm4; z2/+	-	-	p > 0.8	p > 0.6	p < 0.0001
wm4; z2/z2	-	-	-	p > 0.7	p < 0.0001
wm4; CTD	-	-	-	-	p < 0.0001

For each genotype the average relative enrichment from three sets of measurements were compared using a two-tailed Student’s t-test.

Figure 3. ChIP analysis of the white gene in wild-type and w^m4 backgrounds. Histograms of the relative enrichment of chromatin immunoprecipitated by anti-H3S10ph or anti-H3K9me2 antibody from third instar larval salivary glands from wild-type, w^m4 or w^m4 expressing the ΔCTD. For each experimental condition the average relative enrichment normalized to the corresponding control immunoprecipitation from three independent experiments with SD is shown.

Because strong hypomorphic combinations of JIL-1 alleles act as suppressors of PEV (Fig. 2E), the prediction of the model of Lerach et al.⁶ (Fig. 1) is that the relative enrichment of H3K9me2 at the white gene in a w^m4; JIL-1^z2/JIL-1^z2 background should be at or below wild-type levels. JIL-1^z2 is a true null allele¹⁶ without any detectable H3S10 phosphorylation in interphase cells such as third instar salivary gland cells.¹⁵ As illustrated in Figure 4 we found almost no relative enrichment compared with the antibody control ips of both H3K9me2 (1.1 ± 0.1) and H3S10ph (1.1 ± 0.1) in agreement with the above hypothesis. Interestingly, it has been demonstrated that JIL-1^z2 can act as an haplo-enhancer^3,8 (Fig. 2D) and as shown in Figure 4 when only one copy of JIL-1^z2 is present in a w^m4 background the relative enrichment of H3K9me2 (6.7 ± 0.8) increases 6-fold compared with the homozygous JIL-1^z2 condition whereas the relative enrichment of H3S10ph (1.3 ± 0.4) is indistinguishable from that in the w^m4 and w^m4; JIL-1^z2/JIL-1^z2 backgrounds (Tables 1 and 2). Furthermore, when a construct containing only the COOH-terminal domain (CTD) of JIL-1 is expressed in a wild-type background it has a dominant-negative effect and displaces endogenous JIL-1 leading to a striking decrease in histone H3S10ph levels¹⁵ and spreading of H3K9me2 to the chromosome arms as in JIL-1^z2/JIL-1^z2 null mutants.⁵ As illustrated in Figure 4, expression of the CTD in a w^m4 background leads to a relative enrichment of the H3K9me2 (1.4 ± 0.3) and H3S10ph (0.9 ± 0.1) marks indistinguishable from that in the w^m4; JIL-1^z2/JIL-1^z2 background (Tables 1 and 2). Furthermore, this low level of H3K9me2 mark at the white gene is correlated with suppression of PEV (Fig. 2F).⁵

Figure 4. ChIP analysis of the white gene in w^m4 , JIL-1 hypomorphic allelic backgrounds. Histograms of the relative enrichment of chromatin immunoprecipitated by anti-H3S10ph or antiH3K9me2 antibody from third instar larval salivary glands from w^m4; JIL-1^z2/ JIL-1^z2 , w^m4; JIL-1^z2/+ or w^m4 expressing the CTD. For each experimental condition the average relative enrichment normalized to the corresponding control immunoprecipitation from three independent experiments with SD is shown.

Taken together the present experiments indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation providing strong support for the model depicted in Figure 1. In wild type, there are low levels of the epigenetic H3K9me2 mark at the white gene resulting in its normal expression. However, in the w^m4 allele heterochromatic factors can spread across the inversion breakpoint leading to high levels of H3K9me2 at the white gene and silencing of gene expression. Interestingly, we show that this increase in H3K9me2 level can be counteracted by ectopic H3S10 phosphorylation at the white gene restoring gene expression. In contrast, in the absence of H3S10 phosphorylation as it occurs in strong JIL-1 hypomorphic mutant backgrounds there is a redistribution of heterochromatic factors to ectopic chromosome sites resulting in reduced levels of these factors at the pericentric heterochromatin. This leads to less heterochromatic spreading and low levels of H3K9me2 at the white gene in the w^m4 inversion, thus allowing for white gene expression. This expression occurs without H3S10 phosphorylation providing additional evidence that the H3S10 mark is not required for RNA polymerase II-mediated transcription¹⁷ but rather regulates transcription indirectly by counteracting H3K9 dimethylation and gene silencing.

Materials and Methods

Drosophila melanogaster stocks

Fly stocks were maintained at 25°C according to standard protocols.¹⁸ The JIL-1^z2 and the JIL-1^z60 alleles are described in Wang et al.¹⁶ as well as in Zhang et al.¹⁹ The ΔCTD construct containing residue 1–926 and the CTD construct containing sequences from aa 927–1207 in the pYES vector are described in Wang et al.⁵ Expression of the transgenes were driven using a daGAL4 driver introduced by standard genetic crosses. The driver line and the In(1)w^m4 allele was obtained from the Bloomington Stock Center. Balancer chromosomes and markers are described in Lindsley and Zimm.²⁰ w^m4 flies or larvae (w^m4/Y or w^m4) in combination with JIL-1 mutant alleles or expressing the CTD or ΔCTD constructs were generated by standard crossing. Eyes from representative individuals from these crosses were photographed using an Olympus Stereo Microscope and a Spot digital camera (Diagnostic Instruments).

Chromatin immunoprecipitation

For ChIP experiments 50 pairs of salivary glands per sample were dissected from third instar larvae and fixed for 15 min at room temperature in 1 mL of fixative (50 mM HEPES at pH 7.6, 100 mM NaCl, 0.1 mM EDTA at pH 8, 0.5 mM EGTA at pH 8, 2% formaldehyde). Preparation of chromatin for immunoprecipitation was performed as previously described in Legube et al.¹⁴ Rabbit anti-H3S10ph antibody (Active Motif), purified rabbit IgG antibody (Sigma), anti-H3K9me2 mAb (Abcam), or anti-GST mAb 8C7²¹ was used for immunoprecipitation. For each sample the chromatin lysate was divided into equal amounts and immunoprecipitated with experimental and control antibody, respectively. DNA from the immunoprecipitated chromatin fragments (500 bp average) was purified by a Wizard SV DNA purification kit (Promega). The isolated DNA was used as template for quantitative real-time (qRT) PCR performed with the Stratagene Mx4000 real-time cycler. The PCR mixture contained Brilliant II SYBR Green QPCR Master Mix (Stratagene) as well as the corresponding primers: white-forward 5′-GTGCTGTGCCAAAACTCCTC-3′, white-reverse 5′-GATGCTCGGCAGATGGGTTGT-3′ which amplify region -110 to +65. Cycling parameters were 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C. Fluorescence intensities were plotted against the number of cycles using an algorithm provided by Stratagene. DNA levels were quantified using a calibration curve based on dilution of concentrated DNA. For each experimental condition the relative enrichment was normalized to the corresponding control immunoprecipitation from the same chromatin lysate.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.