In this study we show that loss-of-function alleles of the JIL-1 histone H3S10 kinase act as enhancers of position-effect variegation at pericentric sites whereas the gain-of-function JIL-1Su(var)3-1 allele acts as a suppressor strongly supporting a functional role for JIL-1 in maintaining euchromatic chromatin and counteracting heterochromatic spreading and gene silencing.
HIGHER-order chromatin structure is important for epigenetic regulation and control of gene activation and silencing. Transgenes inserted into centromeric regions of chromosomes in Drosophila can exhibit position-effect variegation (PEV), a mosaic silencing of transcription of euchromatic genes as a result of their placement in or near heterochromatin (reviewed by Wallrath 1998; Henikoff 2000; Schotta et al. 2003; Girton and Johansen 2007). It has recently been demonstrated that alleles of the JIL-1 locus are important regulators of chromatin structure and gene expression (Wang et al. 2001; Ebert et al. 2004; Deng et al. 2005; Lerach et al. 2005, 2006). The JIL-1 histone H3S10 tandem kinase localizes specifically to euchromatic interband regions of polytene chromosomes in Drosophila (Jin et al. 1999) and analysis of a JIL-1 null allele, JIL-1z2, has shown that JIL-1 is essential for viability (Wang et al. 2001; Zhang et al. 2003). Furthermore, loss of JIL-1 results in the spreading of the major heterochromatin markers histone H3K9 dimethylation (H3K9me2) and HP1 to ectopic locations on the chromosome arms, and genetic interaction assays have shown that JIL-1 functions antagonistically to Su(var)3-9, which is the major catalyst for dimethylation of the histone H3K9 residue (Schotta et al. 2002; Zhang et al. 2006). On the basis of these findings, Zhang et al. (2006) suggested a model in which JIL-1 histone H3S10 kinase activity functions to maintain euchromatic domains and counteracts heterochromatization and gene silencing. A prediction of this model is that loss-of-function JIL-1 alleles will act as enhancers of PEV resulting in increased silencing of gene expression reporter constructs inserted into normally transcriptionally repressive regions such as pericentric heterochromatin (Johansen and Johansen 2006).
To test this model we examined the effect of decreased levels of JIL-1 protein on expression of a white reporter gene in four P-element transgenic insertion lines (Wallrath and Elgin 1995; Wallrath et al. 1996; Cryderman et al. 1998) (Table 1). Insertion of the P element (P[hsp26-pt, hsp70-w]) into euchromatic sites results in a uniform red eye phenotype whereas insertion into known centromeric heterochromatin regions of the fourth chromosome (line 118E-10), the X chromosome (lines 118E-25 and 118E-32), and the second chromosome (line 39C-3) results in a variegating eye phenotype (Wallrath and Elgin 1995; Wallrath et al. 1996; Cryderman et al. 1998). This silencing within the heterochromatic domains has been correlated with local alterations in chromatin structure and/or shifts in chromatin packaging and suggests a dynamic balance between factors promoting repression and activation of gene expression (Wallrath and Elgin 1995; Cryderman et al. 1998; Sun et al. 2000). Both unique and repetitive DNA sequences were found adjacent to these variegating transgenes, suggesting that PEV does not require that the transgenes be surrounded by repetitive sequences (Cryderman et al. 1998). Furthermore, all of the transgenes show suppression of PEV in response to a mutation in the gene encoding HP1 (Cryderman et al. 1998).
In the experiments the four transgenic reporter lines were crossed into different JIL-1 mutant backgrounds that combined hypomorphic and null JIL-1 alleles (JIL-1z60 and JIL-1z2) to generate progeny expressing decreased amounts of wild-type JIL-1 protein. The JIL-1z60 allele is a strong hypomorph producing only 0.3% of wild-type JIL-1 protein levels, whereas the JIL-1z2 allele is a true null and homozygous animals do not survive to adulthood (Wang et al. 2001; Zhang et al. 2003). The JIL-1z2/JIL-1z60 heteroallelic combination is semilethal and only a limited number of eclosed animals from large-scale crosses could be analyzed. In addition, we compared the effect of the loss-of-function JIL-1 alleles to that of the dominant gain-of-function JIL-1Su(var)3-1 allele that is one of the strongest suppressors of PEV so far described (Ebert et al. 2004). The JIL-1Su(var)3-1 allele generates truncated proteins with COOH-terminal deletions that mislocalize to ectopic chromosome sites (Ebert et al. 2004; Zhang et al. 2006). Flies from each of the four transgenic lines with the different JIL-1 genotypes were scored for the percentage of the eye that had red ommatidia and compared to flies containing wild-type levels of JIL-1 protein (Figures 1 and 2 and Table 2). Although both male and female flies were scored, due to sex differences results from only female flies are shown. However, the trend observed in male flies was identical to that in female flies. As illustrated in Figure 1, hypomorphic allelic combinations of the JIL-1 alleles JIL-1z60 and JIL-1z2 lead to a strong enhancement of PEV as indicated by the nearly completely white eye phenotype, whereas in contrast, the homozygous gain-of-function JIL-1Su(var)3-1 allele leads to strong suppression of PEV as indicated by the nearly completely red eye phenotype. The effect of the JIL-1 alleles on the distribution of the proportion of red ommatidia in the four centromeric P-element insertion lines is shown in Figure 2 and Table 2. As indicated in Figure 2, the strongly hypomorphic allelic combination of JIL-1z2/JIL-1z60 leads to a greater enhancement of PEV than the less severe JIL-1z60/JIL-1z60 allelic combination. In addition, we compared the mean proportion of the eyes that had red ommatidia between JIL-1z2/JIL-1z60, +/+, and JIL-1Su(var)3-1/JIL-1Su(var)3-1 flies, respectively, for each of the four transgenic lines using a Student's t-test. In each case the mean proportions were significantly different (P < 0.01). Thus, these results show that while loss-of-function JIL-1 alleles act as enhancers of PEV at pericentric sites the gain-of-function JIL-1Su(var)3-1 allele acts as a suppressor of PEV strongly supporting the model for JIL-1's function in counteracting heterochromatic spreading and gene silencing.
Interestingly, it has previously been demonstrated that both JIL-1 hypomorphic loss-of-function mutations and gain-of-function JIL-1Su(var)3-1 alleles act as strong suppressors of PEV of the wm4 allele (Ebert et al. 2004; Lerach et al. 2006). The In(1)wm4 X chromosome contains an inversion that juxtaposes the euchromatic white gene and heterochromatic sequences adjacent to the centromere (Muller 1930; Schultz 1936). Studies of PEV of this allele suggest that the degree of silencing may depend on the amount of heterochromatic factors at the breakpoint (reviewed in Weiler and Wakimoto 1995; Girton and Johansen 2007). In this model a reduction in the amount of these factors in centromeric regions would limit heterochromatic spreading over long distances but would be predicted to not affect short range silencing. Thus, the finding that both JIL-1 hypomorphic loss-of-function mutations and gain-of-function JIL-1Su(var)3-1 alleles act as suppressors of PEV of the wm4 allele, whereas they have opposite effects on PEV of transgenes inserted directly into centromeric heterochromatin, supports this model. In the case of wm4 the redistribution of the major heterochromatin markers H3K9me2 and HP1 to ectopic locations on the chromosome arms and the resulting decrease in the concentration of these factors at centromeric regions occurring in JIL-1 loss-of-function mutants (Zhang et al. 2006) diminishes the ability of pericentric heterochromatin to spread long distances, leading to a reduction of wm4 silencing (Lerach et al. 2006). In contrast, the results of this study indicate that in the absence of JIL-1 histone H3S10 kinase activity transcription of euchromatic transgenes in direct proximity to pericentric heterochromatin is almost completely suppressed.
We thank members of the laboratory for discussions and critical reading of the manuscript. We also wish to acknowledge V. Lephart for maintenance of fly stocks and Laurence Woodruff for technical assistance. We especially thank L. Wallrath and D. Cryderman for providing the P-element insertion lines and for helpful advice. This work was supported by NIH Grant GM62916 (K.M.J.).
Communicating editor: J. A. Birchler
- Received March 20, 2007.
- Accepted April 12, 2007.
- Copyright © 2007 by the Genetics Society of America