JIL-1 and Su(var)3-7 interact genetically and counteract each other's effect on position-effect variegation in Drosophila

Abstract

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity.

Figure 1.—

Morphology of polytene chromosomes in JIL-1 and Su(var)3-7 double mutant backgrounds. Polytene chromosome preparations from third instar male and female larvae were labeled with Hoechst to visualize the chromatin. Note the misalignment and intermixing of interband and banded regions and the extensive coiling and folding of the chromosome arms in JIL-1^z2/JIL-1^z2 (z2/z2) mutant chromosomes as compared to wild type (wt). The male X chromosome (X) was particularly affected and no remnants of banded regions were discernible. In JIL-1 and Su(var)3-7 double mutant backgrounds from male and female JIL-1^z2 Su(var)3-7^7.1A/JIL-1^z2 (z2, 3-7^7.1A/z2) and JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2 (z2, 3-7¹⁴/z2) larvae, the polytene chromosome morphology was indistiguishable from that of JIL-1^z2/JIL-1^z2 homozygous null mutants.

Figure 2.—

Localization of H3K9me2 in polytene chromosomes from JIL-1 and Su(var)3-7 mutant female third instar larvae. The polytene squash preparations were labeled with antibody to H3K9me2 (in red) and with Hoechst (DNA, in blue/gray). The X chromosome is indicated by an X. Preparations from wild-type (wt), heterozygous JIL-1^z2/+ (z2/+), homozygous JIL-1^z2/JIL-1^z2 (z2/z2), JIL-1^z2 Su(var)3-7^7.1A/JIL-1^z2 (z2, 3-7^7.1A/z2), JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2 (z2, 3-7¹⁴/z2), JIL-1^z2 Su(var)3-7^R2a8/JIL-1^z2 (z2, 3-7^R2a8/z2), and JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2 Su(var)3-7¹⁴ (z2, 3-7¹⁴/z2, 3-7¹⁴) larvae are shown. In wild-type and JIL-1^z2/+ preparations, H3K9me2 labeling was mainly localized to and abundant at the chromocenter; however, in the absence of the JIL-1 kinase, the H3K9me2 labeling spread to the autosomes and particularly to the X chromosome (see also Zhang et al. 2006; Deng et al. 2007). In JIL-1^z2 Su(var)3-7^7.1A/JIL-1^z2, JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2, and JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2 Su(var)3-7¹⁴ double mutant larvae, the H3K9me2 labeling was indistiguishable from that of JIL-1^z2/JIL-1^z2 homozygous null mutants.

Figure 3.—

Localization of Su(var)3-7 in polytene chromosomes from JIL-1, Su(var)3-7, and Su(var)3-9 mutant female third instar larvae. The polytene squashes were labeled with antibody to Su(var)3-7 (in green) and with Hoechst (DNA, in blue/gray). The chromocenter is indicated with an asterisk and n indicates weak background labeling of the nucleolus in some of the preparations. Preparations from wild-type, JIL-1^z2 homozygous (z2/z2), JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2 (z2, 3-7¹⁴/z2), and Su(var)3-9⁰¹/Su(var)3-9⁰² (3-9⁰¹/3-9⁰²) larvae are shown. In wild-type preparations, Su(var)3-7 labeling was mainly localized to and abundant at the chromocenter; however, in the absence of the JIL-1 kinase, the Su(var)3-7 labeling spread to the chromosome arms with a concomitant decrease at the chromocenter. In JIL-1^z2 Su(var)3-7¹⁴/JIL-1^z2 and Su(var)3-9⁰¹/Su(var)3-9⁰² mutant larvae Su(var)3-7 labeling was greatly reduced and mainly confined to the chromocenter.

Figure 4.—

Counterbalancing effect of JIL-1 and Su(var)3-7 loss-of-function alleles on PEV of the P-element insertion line 118E-15. (A) Examples of the degree of PEV in the eyes of wild-type JIL-1 and Su(var)3-7 (wt), JIL-1^z60/JIL-1^z2 (z2/z60), Su(var)3-7^7.1A/+, and JIL-1^z2 Su(var)3-7^7.1A/JIL-1^z60 (z2, 3-7^7.1A/z60) flies in a 118E-15 background. All images are from male flies. (B) Histograms of the distribution of the percentage of red ommatidia in wt, JIL-1^z60/JIL-1^z2 (z2/z60), Su(var)3-7^7.1A/+, and JIL-1^z2 Su(var)3-7^7.1A/JIL-1^z60 (z2, 3-7^7.1A/z60) male flies homozygous for 118E-15.

Figure 5.—

Counterbalancing effect of JIL-1 and Su(var)3-7 loss-of-function alleles on PEV in the eyes of w^m4 flies. (A) Examples of the degree of PEV in the eyes of wild-type JIL-1 and Su(var)3-7 (wt), JIL-1^z2/+ (z2/+), Su(var)3-7^7.1A/+, and JIL-1^z2 Su(var)3-7^7.1A/+ (z2, 3-7^7.1A/+) flies in a w^m4 background. All images are from male flies. (B) Histograms of the distribution of the percentage of red ommatidia in wt, JIL-1^z2/+ (z2/+), Su(var)3-7^7.1A/+, and JIL-1^z2 Su(var)3-7^7.1A/+ (z2, 3-7^7.1A/+) male flies in a w^m4 background.

Figure 6.—

Counterbalancing effect of JIL-1 and Su(var)3-7 loss-of-function alleles on PEV in the eyes of DX1 flies. (A) Examples of the degree of PEV in the eyes of wild-type JIL-1 and Su(var)3-7 (wt), JIL-1^z2/+ (z2/+), Su(var)3-7¹⁴/+, and JIL-1^z2 Su(var)3-7¹⁴/+ (z2, 3-7¹⁴/+) flies in a DX1 background. All images are from male flies. (B) Histograms of the distribution of the percentage of red ommatidia in wt, JIL-1^z2/+ (z2/+), Su(var)3-7¹⁴/+, and JIL-1^z2 Su(var)3-7¹⁴/+ (z2, 3-7¹⁴/+) male flies in a DX1 background.