THE JIL-1 KINASE, A MEMBER OF THE MSL COMPLEX, IS NECESSARY FOR PROPER DOSAGE COMPENSATION OF EYE PIGMENTATION IN DROSOPHILA

Abstract

The upregulation of the JIL-1 kinase on the male X chromosome and its association with the MSL complex suggest that JIL-1 may play a role in regulating dosage compensation. To directly test this hypothesis we measured eye pigment levels of mutants in the X-linked white gene in an allelic series of JIL-1 hypomorphic mutants. We show that dosage compensation of w^a alleles that normally do exhibit dosage compensation was severely impaired in the JIL-1 mutant backgrounds. As a control we also examined a hypomorphic white allele w^e that fails to dosage compensate in males due to a pogo element insertion. In this case the relative pigment level measured in males as compared to females remained approximately the same even in the most severe JIL-1 hypomorphic background. These results indicate that proper dosage compensation of eye pigment levels in males controlled by X-linked white alleles requires normal JIL-1 function.

We have recently characterized a novel tandem kinase in Drosophila, JIL-1, that associates with the chromosomes throughout the cell cycle, localizes specifically to the gene-active interband regions of larval polytene chromosomes, and phosphorylates histone H3S10 (Jin et al., 1999; Wang et al., 2001; Zhang et al., 2003). Analysis of JIL-1 null and hypomorphic alleles showed that JIL-1 is essential for viability and that reduced levels of JIL-1 protein lead to a global change in chromosome structure (Wang et al., 2001; Deng et al., 2005). However, JIL-1 is also found at two-fold higher levels on the male X chromosome and associates with the male specific lethal (MSL) dosage compensation complex (Jin et al., 2000). The MSL complex is required for the necessary hypertranscription of genes on the male X chromosome for dosage compensation in flies (reviewed in Meller & Kuroda, 2002). This enhanced transcription is thought to arise from MSL complex-induced histone H4 acetylation generating a more open chromatin structure (Smith et al., 2000). The upregulation of JIL-1 protein on the male X chromosome, JIL-1’s association with the MSL complex, and the higher level of male lethality associated with hypomorphic JIL-1 alleles (Wang et al., 2001) all suggest that JIL-1 may play a role in regulating dosage compensation. However, so far evidence for this hypothesis has only been correlative. Therefore, to directly examine whether JIL-1 is involved in male upregulation of genes whose increased expression levels depend upon the dosage compensation complex we have compared the eye pigment levels controlled by X-linked white alleles in males and females in different JIL-1 hypomorphic mutant backgrounds.

In these studies we have taken advantage of the properties of two alleles of the white locus, white[apricot] (w^a) and white[eosin] (w^e). The w^a allele was created by the insertion of a copia transposon that results in an overall reduction of red pigment in the eyes of both male and female flies (Zachar & Bingham, 1982). Males carrying the w^a allele retain the ability to dosage compensate for the white gene, and they show roughly equivalent expression to that of the female carrying two copies of w^a (Zachar & Bingham, 1982; this study). This is in contrast to the hypomorphic w^e allele which has lost the ability to dosage compensate in males (Smith & Lucchesi, 1969). This mutation is due to insertion of a pogo element into the Doc element present in the w¹ allele (O’Hare et al., 1991). The w^a and w^e alleles were chosen for this study because they are both hypomorphic alleles with comparable pigment levels and because their eye pigments have maximum absorbance at the same wavelength. In the experiments the w^a and w^e alleles were crossed into different JIL-1 mutant backgrounds that combined hypomorphic and null JIL-1 alleles (JIL-1^z28, JIL-1^z60, and JIL-1^z2) in order to generate progeny producing different amounts of JIL-1 protein. The JIL-1^z28 allele is a weak hypomorph producing 45% the normal level of wild-type JIL-1 protein, the JIL-1^z60 allele is a strong hypomorph producing only 0.3% of wild-type JIL-1 protein levels, whereas the JIL-1^z2 allele is a true null and homozygous animals do not survive to adulthood (Wang et al., 2001; Zhang et al., 2003). The JIL-1^h9 allele expresses a truncated JIL-1 protein that lacks part of the second kinase domain and the entire COOH-terminal domain and acts as a strong hypomorph (Zhang et al., 2003). The JIL-1^z60/JIL-1^z60 and JIL-1^z2/JIL-1^z60 allelic combinations are semi-lethal and only a few eclosed animals from large scale crosses could be analyzed. To compare eye pigment levels between male and female flies we performed pigment assays essentially as in Ashburner (1989) using sets of 10 pooled fly heads of each genotype. It should be noted that the z series of JIL-1 mutations carry the ry⁵⁰⁶ allele which modifies pigment color (Rørth et al., 1998) wherefore the optical density measurements cannot be directly compared between these alleles, wild-type, and heteroallelic combinations with JIL-1^h9. However, this difference does not affect the determination of the relative optical density of pigment levels in males and females of the same genotype.

The results show that as JIL-1 levels decrease in an allelic series of JIL-1 mutants the male to female pigment ratio of w^a eye pigment decreases (Figure 1A and Table 1). In a wild-type JIL-1 background the pigment ratio was 1.00; however, this ratio was only 0.64 in the most severe hypomorphic JIL-1 heteroallelic combination (JIL-1^z2/JIL-1^h9). This difference was statistically significant (p<0.005, Student’s t-test). Thus, these results indicate that males were unable to properly dosage compensate in the absence of normal levels of JIL-1 protein. However, that the ratio was not reduced to 50% suggests that partial dosage compensation can still occur possibly due to the low level of JIL-1 activity that was necessary to allow eclosion or the presence of a parallel pathway independent of JIL-1. Interestingly, consistent with previous observations of a clear maternal effect of JIL-1 (Zhang et al., 2003) dosage compensation was most affected in JIL-1 mutant heteroallelic combinations where the most severely hypomorphic allele was maternally provided (Table 1).

Figure 1.

JIL-1 regulates dosage compensation at the white locus in males. (A) The ratio of male/female eye pigment levels in an allelic series of JIL-1 hypomorphs (comprised of the alleles JIL-1^z28, JIL-1^z60, JIL-1^h9, JIL-1^z2) in a w^a mutant background. In w^a flies a copia retrotransposon causes a general reduction of pigment level in both sexes without interfering with the dosage compensation process. The decrease in male/female eye pigment ratios correlates directly with the severity of the JIL-1 hypomorphic alleles. (B) In w^e mutant flies there is no dosage compensation due to a pogo element insertion. In this mutant background there is no significant effect of JIL-1 hypomorphic alleles on male/female eye pigment ratios. Each histogram represents the average male/female pigment ratio from sets of OD₃₆₅ measurements from 10 pooled fly heads with S.D. The average male/female pigment ratio in JIL-1 mutants were compared to that in a wild-type JIL-1 background using a Student’s t-test.

Table 1.

Dosage compensation of eye pigment in w^a flies in JIL-1 mutant backgrounds

genotypea	sex	nb	OD365 with S.D	male/female pigment ratio	Student’s t-test
+/+	female	5	0.416±0.033	1.00
+/+	male	5	0.414±0.022
z28/z28	female	3	0.220±0.018	0.98	p>0.35
z28/z28	male	3	0.216±0.028
z2/z28	female	3	0.254±0.041	0.81	p<0.01
z2/z28	male	3	0.205±0.016
h9/z2	female	6	0.441±0.019	0.71	p<0.005
h9/z2	male	6	0.315±0.020
z60/z60	female	2	0.224±0.046	0.69	p<0.05
z60/z60	male	2	0.154±0.016
z60/z2	female	4	0.312±0.027	0.68	p<0.005
z60/z2	male	4	0.212±0.012
z2/h9	female	10	0.438±0.020	0.64	p<0.005
z2/h9	male	10	0.279±0.019

^aGenotype of the third chromosome with maternal contribution listed first. In addition, all flies are homozygous (females) or hemizygous (males) for w^a on the X chromosome. The z series of JIL-1 mutations also carry the ry⁵⁰⁶ allele.

^bEach pigment concentration was determined from 10 pooled fly heads.

It has been demonstrated that the effect of loss of JIL-1 on chromatin structure of the male X chromosome is qualitatively different from that of the autosomes and the female X chromosome (Deng et al., 2005). Therefore, to control for effects of the JIL-1 hypomorphic mutations specific to the male X chromosome, but unrelated to dosage compensation mechanisms, we examined the male/female pigment ratio in the eyes of w^e flies. As shown in Figure 1B and Table 2 w^e males do not dosage compensate and have only about half (46%) the pigment level of w^e females (see also Smith & Lucchesi, 1969). This ratio was not significantly changed in hypomorphic JIL-1 mutant backgrounds (Figure 1B and Table 2) suggesting that JIL-1 did not have a differential effect on white expression in males as compared to females unrelated to the function of the dosage compensation machinery. Furthermore, these data suggest that while the optical density measurements of pigment expression levels varied somewhat in the different mutant backgrounds this did not affect the determination of the male/female pigmentation ratios in flies of the same genotype. Thus, our results strongly indicate that JIL-1 function is required for proper dosage compensation at the white locus in Drosophila. In future experiments it will be informative to further explore how the interaction between JIL-1 and the MSL complex may contribute to regulation of the dosage compensation machinery.

Table 2.

Dosage compensation of eye pigment in w^e flies in JIL-1 mutant backgrounds

genotypea	sex	nb	OD365 with S.D.	male/female pigment ratio	Student’s t-test
+/+	female	5	0.738±0.080	0.46
+/+	male	5	0.340±0.143
z2/z28	female	5	0.361±0.030	0.49	p>0.20
z2/z28	male	5	0.176±0.009
z60/z60	female	1	0.241	0.43
z60/z60	male	1	0.104
z2/h9	female	6	0.376±0.030	0.45	p>0.30
z2/h9	male	6	0.168±0.029

^aGenotype of the third chromosome. In addition, all flies are homozygous (females) or hemizygous (males) for w^e on the X chromosome. The z series of JIL-1 mutations also carry the ry⁵⁰⁶ allele.

^bEach pigment concentration was determined from 10 pooled fly heads.

MATERIALS AND METHODS

Drosophila stocks and crosses

Fly stocks were maintained according to standard protocols (Roberts, 1998). Oregon-R was used for wild-type preparations. The JIL-1^z2, JIL-1^z28, JIL-1^z60, and JIL-1^h9 alleles are described in Wang et al. (2001) and in Zhang et al. (2003). The z series of JIL-1 mutations carry the ry⁵⁰⁶ allele (Rørth et al., 1998). Balancer chromosomes and markers are described in Lindsley & Zimm (1992). Strains containing either the w^a or the w^e allele on the X chromosome and a loss-of-function JIL-1 allele (either JIL-1^z2, JIL-1^z60, JIL-1^z28, or JIL-1^h9) heterozygous with the TM6 Sb Tb e third chromosome balancer were produced by standard crossing. Subsequent crosses between these strains generated flies with different JIL-1 allelic combinations in w^a or w^e backgrounds.

Eye pigment assays

For pigment level comparisons adult flies were collected from the respective crosses at eclosion, aged 6 days at 25°C, frozen in liquid nitrogen, and stored at −80°C until assayed. The pigment assays were performed essentially as in Ashburner (1989) using sets of 10 fly heads of each genotype collected from males and females, respectively. For each sample the heads from the 10 flies were homogenized in 125 μl of chloroform and 125 μl 0.1% ammonium hydroxide, centrifuged, and the optical density (OD) of the supernatant spectrophotometrically measured at a wavelength of 365 nm. In order to obtain a measure of the relative pigments levels in male and female eyes the OD determined for male flies was divided by the OD determined for female flies of the same genotype.