Multiple SET methyltransferases are required to maintain normal heterochromatin domains in the genome of Drosophila melanogaster

Abstract

Methylation of histone H3 lysine 9 (H3K9) is a key feature of silent chromatin and plays an important role in stabilizing the interaction of heterochromatin protein 1 (HP1) with chromatin. Genomes of metazoans such as the fruit fly Drosophila melanogaster generally encode three types of H3K9-specific SET domain methyltransferases that contribute to chromatin homeostasis during the life cycle of the organism. SU(VAR)3-9, dG9a, and dSETDB1 all function in the generation of wild-type H3K9 methylation levels in the Drosophila genome. Two of these enzymes, dSETDB1 and SU(VAR)3-9, govern heterochromatin formation in distinct but overlapping patterns across the genome. H3K9 methylation in the small, heterochromatic fourth chromosome of D. melanogaster is governed mainly by dSETDB1, whereas dSETDB1 and SU(VAR)3-9 function in concert to methylate H3K9 in the pericentric heterochromatin of all chromosomes, with dG9a having little impact in these domains, as shown by monitoring position effect variegation. To understand how these distinct heterochromatin compartments may be differentiated, we examined the developmental timing of dSETDB1 function using a knockdown strategy. dSETDB1 acts to maintain heterochromatin during metamorphosis, at a later stage in development than the reported action of SU(VAR)3-9. Surprisingly, depletion of both of these enzymes has less deleterious effect than depletion of one. These results imply that dSETDB1 acts as a heterochromatin maintenance factor that may be required for the persistence of earlier developmental events normally governed by SU(VAR)3-9. In addition, the genetic interactions between dSETDB1 and Su(var)3-9 mutations emphasize the importance of maintaining the activities of these histone methyltransferases in balance for normal genome function.

Figure 1.—

In contrast with dG9a, dSETDB1 is a major H3K9 methyltransferase that is required for heterochromatin formation in Drosophila. (A) H3K9 methyltransferases differ in their expression patterns during Drosophila development. RT–PCR assays with primer sets specific for each methyltransferase sequence are shown in the top three panels; RpL32 amplification, both with and without reverse transcriptase, is provided at the bottom as an input control. All samples are from wild-type OR flies. Embryos, 6- to 18-hr mixed embryos; larvae, third instar. (B) dSETDB1, dG9a, and SU(VAR)3-9 all participate in methyl-H3K9 metabolism in vivo. Duplicate Western blots of wild-type vs. HMT mutant lysates from adult flies were probed with antibodies specific for mono-, di- and trimethyl H3K9; anti-H2B was used as loading control. Knockdown was achieved by using the Act5CGAL4 driver with dSETDB1hp^2101B or dG9ahp¹⁰⁰². Numbers at the bottom of each blot represent the normalized band intensity expressed as fold difference from wild type. (C) dSETDB1^KD is a potent suppressor of variegation, while dG9a^KD is not. PEV assays shown compare Mod(var) effects of dG9a^KD vs. dSETDB1^KD using representative fourth chromosome (39C-12) and pericentric (118E-28) reporters. Knockdown was achieved by using the daGAL4 driver with dSETDB1hp^2101B or dG9ahp¹⁰⁰². To the right of the eye images, data from quantitative eye pigment assays are shown, confirming the Su(var) effect. Shown in light gray are values from flies carrying the indicated hairpin construct and the reporter in the absence of the driver, while the dark gray bars illustrate the pigment level in flies that carry the reporter, the hairpin construct, as well as the driver. Y-axis, fraction of wild-type pigment. Error bars represent standard deviations.

Figure 2.—

The knockdown system verifies that dSETDB1 and SU(VAR)3-9 have opposing roles in fourth chromosome heterochromatin formation, but act similarly in pericentric heterochromatin. (A) dSETDB1^KD (5–25% of wild-type expression, see supplemental Figure S1) is a suppressor of variegation for insertions throughout the Drosophila genome. Su(var)3-9⁰⁶ is a dominant suppressor of pericentric variegation on all chromosomes including the fourth but acts as a dominant enhancer of variegation on reporters in the banded region of the fourth chromosome. For the dSETDB1 knockdown the daGAL4 driver was combined with dSETDB1hp^2101B. (B) Quantification of pigment content of all populations represented in A. Values for the “parent” control are shown in lightest shade of gray, for Su(var)3-9⁰⁶ lines in the medium shade of gray, and for dSETDB1^KD lines in dark gray. Note the distinct E(var) effect of Su(var)3-9⁰⁶ at variegating reporters in the arm of the fourth chromosome, and the large Su(var) effect of dSETDB1^KD at all sites (refer to Table 3 for numerical values). Error bars are standard deviation (n = 3). Parent refers to the reporter in a yw background.

Figure 3.—

A complex interaction between loss of dSETDB1 and of SU(VAR)3-9 impacts heterochromatin formation, with the double mutant showing a less severe phenotype. (A) dSETDB1 and dG9a act with SU(VAR)3-9 to produce methyl-H3K9 in vivo. Western blot showing quantities of H3K9 methylation in double mutants (adults). Values at the bottom of each blot express the fraction of mono-, di-, or trimethylation in double mutants by comparison with homozygous Su(var)3-9⁰⁶. Knockdown of dG9a and dSETDB1 was achieved by combining the Act5CGAL4 driver with hairpins dG9ahp²²⁰¹ or dSETDB1hp⁰⁴⁰⁸. (B) Loss of silencing in dSETDB1^KD lines is partially restored by depletion of SU(VAR)3-9, except at pericentric fourth chromosome heterochromatin. Pigment values for dominant suppression by Su(var)3-9⁰⁶/+, dSETDB1^KD and dSETDB1^KD;Su(var)3-9⁰⁶/+ are compared with parental (no HMT mutant) values. Knockdown of dSETDB1 was achieved by combining the daGAL4 driver with hairpin dSETDB1hp^2101B. Error bars represent standard deviations (n = 3). (C) Comparison of fold suppression changes from the parental pigment values are expressed for single and double mutants on the basis of the data from B. Enhancement of PEV (reduced pigmentation) is expressed as a negative fold change.

Figure 4.—

The fourth chromosome appears hypercondensed in Su(var)3-9 mutant salivary glands; this is reversed in Su(var)3-9; dSETDB1 double mutants. (A) Localization of heterochromatin components on polytene chromosomes from wild-type, Su(var)3-9⁰⁶ homozygous mutant, dSETDB1 mutant (dSETDB1hp^2101B driven by daGAL4), and egg¹⁴⁷³;Su(var)3-9⁰⁶ double homozygous mutant chromosomes. Retention of H3K9me₃ (green in merged image) and superabundance of HP1 (red) on the fourth chromosome (asterisk) is observed in Su(var)3-9⁰⁶ mutants. A broader distribution of HP1 is observed in egg¹⁴⁷³;Su(var)3-9⁰⁶ double-mutant salivary glands. (B) Heterozygosity for egg²³⁵ reduces condensation of chromosome four in Su(var)3-9⁰⁶ mutants. Polytene chromosomes comparing HP1 localization in wild-type, Su(var)3-9⁰⁶, and egg²³⁵/+;Su(var)3-9⁰⁶ lines. Close-up views of the chromocenter are provided at the right of each image showing each set of chromosomes with the fourth chromosome indicated by an asterisk. Side-by-side comparisons (generated by squashing the different salivary glands together on the same slide) are provided in supplemental Figure S3.

Figure 5.—

dSETDB1 contributes to fourth chromosome heterochromatin formation during metamorphosis. (A) Schematic illustration of drivers spanning the development of Drosophila used to assess impact of dSETDB1 knockdown on PEV at different developmental times. (B) dSETDB1 is required for heterochromatin formation and/or maintenance after oogenesis but prior to late pupal development. Representative eye phenotypes resulting from knockdown of dSETDB1 in the ovary and early embryo (nosGAL4) are contrasted with parental phenotypes and ubiquitous knockdown of this methyltransferase enzyme (daGAL4). Knockdown of dSETDB1 in eye tissue lineages (eyGAL4) results in an unusual pattern of suppression. [Note that unlike the other drivers, eyGAL4 is marked with a copy of white; this results in the low level background pigmentation shown (1Xey, no hp) but does not interfere with the assay.] dSETDB1hp^2101B was used for these experiments. The reporter was line 39C-12 (insertion in the banded region of chromosome 4) or 118E-10 (insertion in the pericentric heterochromatin, chromosome 4) as indicated. (C) The wedge-shaped pattern of hsp70-w expression (depicted at left; 118E-10) displayed when knockdown is driven by 1XeyGAL4 indicates that dSETDB1 is specifically required for the stability of heterochromatin during morphogenetic furrow (MF) progression in the eye imaginal disc (illustrated at right), which occurs during late larval development. D, dorsal; V, ventral; A, anterior; and P, posterior.

Figure 6.—

Global changes in gene expression occur in histone methyltransferase mutant Drosophila. (A) Venn diagram illustrating overlap of misregulated genes in Su(var)3-9⁰⁶ (red) and egg²³⁵/+ (blue) single mutants as well as the egg²³⁵/+; Su(var)3-9⁰⁶ (yellow) double mutant. At the bottom of each genotype, the total number of misregulated genes detected is given. RNA prepared from fly heads. (B) Bar chart depicting the percentage of misregulated genes in each mutant background compared to wild type by chromosome. A high proportion of fourth chromosome genes are misregulated as a consequence of the dSETDB1 mutation. (C) Bar chart depicting the percentage of misregulated genes in each heterochromatin domain. (D) Bar chart illustrating transposon reactivation in each mutant background based on increased expression levels compared to wild type. Loss of SU(VAR)3-9 activity has the more pronounced effect on transposon expression; this is frequently reversed in the double-mutant line.