Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing

Abstract

Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila. SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7. Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.

Fig. 1. Distribution of the SU(VAR)3–9 protein in salivary gland polytene chromosomes. (A and B) Immunostaining of polytene chromosomes from a pP{GS[ry⁺, hs (Su(var)3–9 cDNA)–EGFP]} transgenic line with a rabbit polyclonal α-GFP antibody. (C and D) Heterochromatin association of endogenous SU(VAR)3–9 after staining with a rabbit polyclonal α-SU(VAR)3–9 antibody. DNA staining with propidium iodide (A and C) and immunostaining of the same nuclei, respectively (B and D). (E) Western blot of nuclear extracts from wild-type and Su(var)3–9 null mutant embryos. Blot probed with α-SU(VAR)3–9 antibody (upper panel) and α-HP1 antibody (lower panel). (F–H) Confocal sections of salivary gland nuclei showing in vivo distribution of SU(VAR)3–9–EGFP in wild-type (F), T(2;3)TE80var20/+; pP{GS[ry⁺, hs (Su(var)3–9 cDNA)–EGFP]} (G) and w^m4/w^m4; pP{GS[ry⁺, hs (Su(var)3–9 cDNA)–EGFP]} (H) larvae. The chromosomal rearrangements relocate regions of heterochromatin to distal euchromatin (arrows).

Fig. 2. Histone H3-K9 methylation in heterochromatin is controlled by SU(VAR)3–9. (A and B) H3-K9 HMTase activity of recombinant SU(VAR)3–9 protein for histone H3 (A) and N-terminal peptide (1–20) of histone H3 (B) compared with wild-type and mutant (H324L) human SUV39H1. Arrowheads mark the recombinant proteins. (C) Western analysis of nuclear extracts from wild-type and Su(var)3–9 null mutant [Su(var)3–9⁰⁶/Su(var)3–9⁰⁶] embryos with an α-dimethyl H3-K9 antibody. (D) Immunostaining of wild-type larval salivary gland chromosomes with α-dimethyl H3-K9 antibody. Whole chromosome set and enlargement of chromocentre region. Arrows denote the fourth chromosome. (E) Immuno staining of SU(VAR)3–9-deficient [Su(var)3–9⁰⁶/Su(var)3–9⁰⁶] larvae with α-dimethyl H3-K9 antibody. Arrows in enlarged chromocentre denotes the fourth chromosome. (F) Reconstitution of H3-K9 methylation in chromocentre heterochromatin by expression of SU(VAR)3–9–EGFP in Su(var)3–9 null mutant larvae. DNA staining with propidium iodide (D and E) and TOTO-3 (F).

Fig. 3. Yeast two-hybrid analysis of SU(VAR)3–9 interaction with HP1 and SU(VAR)3–7. Two-hybrid interaction tests of SU(VAR)3–9 baits with HP1 and SU(VAR)3–7 target constructs. LacZ activities are shown in parentheses. SU(VAR)3–9 regions are indicated, shaded for the overlapping 81 amino acid region with eIF2γ: yellow for the chromodomain, dark green for the SAC domain and green for the SET domain. In HP1 the chromodomain is indicated in yellow and the chromo-shadow domain in amber. Zinc fingers in SU(VAR)3–7 are indicated by white stripes.

Fig. 4. In vivo and cytological analysis of interaction between SU(VAR)3–9 and HP1. (A and B) Immunoprecipitation of SU(VAR)3–9–EGFP (A) and HP1 (B) from heat-shocked transgenic and untreated wild-type 0–12 h embryos, respectively. Blots were probed with (A) α-SU(VAR)3–9, and (B) α-GFP (upper panels) and α-HP1 (lower panels). Ex, nuclear extract; Sn, supernatant; IP, immunoprecipitate. Arrowheads mark the signal of the antibody used for co-immunoprecipitation. (C) In vivo relocation of SU(VAR)3–9–EGFP by an HP1/PC chimeric protein. GFP fluorescence in a confocal section of a salivary gland nucleus. (D) Immunocytological analysis of transheterozygous larvae expressing SU(VAR)3–9–EGFP and the HP1/PC chimeric protein with α-GFP for SU(VAR)3–9–EGFP and α-β-gal for HP1/PC. (E) Double immunostaining for SU(VAR)3–9–EGFP with a mouse monoclonal α-GFP antibody and with a rabbit polyclonal α-HP1 antibody.

Fig. 5. Control of SU(VAR)3–9 and HP1 heterochromatin association. (A) Confocal sections of unfixed larval salivary gland nuclei expressing full-length SU(VAR)3–9–EGFP, SU(VAR)3–9NTerm–EGFP, SU(VAR)3–9Δchromo–EGFP, SU(VAR)3–9ΔSET–EGFP and SU(VAR)3–9TRX–EGFP protein variants in SU(VAR)3–9-deficient larvae. Arrows point to the fourth chromosome. (B) In vivo distribution of SU(VAR)3–9–EGFP in wild-type and HP1 deficient [Df(2L)TE128 × 11/Su(var)2–5⁰⁴] salivary gland nuclei. (C) α-dimethyl H3-K9 antibody staining of an HP1-deficient salivary gland nucleus. DNA staining with propidium iodide. (D) In vivo analysis of HP1–EGFP heterochromatin association in wild-type and SU(VAR)3–9-deficient [Su(var)3–9⁰⁶/Su(var)3–9⁰⁶] nuclei of third instar larval salivary glands. Arrows point to the fourth chromosome.

Fig. 6. Genetic interactions between Su(var)3–9, Su(var)2–5 and Su(var)3–7 and the modifier effect of Y chromosome aneuploidy in w^m4 PEV. (A) Heterozygotes for the loss-of-function mutant alleles Su(var)3–9⁰⁶, Su(var)2–5⁰⁵ and Df(3R)AceD1[Su(var)3–7 null] were combined with transgenic constructs introducing extra genomic copies of Su(var)3–9, Su(var)2–5 or Su(var)3–7, respectively. Resultant white variegation in offspring w^m4 female is shown. (B) Genetic interaction to the PEV modifier effect of Y chromosome aneuploidy. First row shows white variegation in w^m4/O males of +/+ (control) and of heterozygotes of the loss-of-function Su(var)3–9⁰⁶, Su(var)2–5⁰⁵ and Df(3R)AceHD1[Su(var)3–7 null] alleles, respectively. Second row shows white variegation in w^m4/w/Y females of +/+ (control) and of heterozygotes with an additional genomic copy of Su(var)3–9, Su(var)2–5 and Su(var)3–7, respectively.

Fig. 7. Expression of human SUV39H1 in Drosophila. (A) Association of SUV39H1–EGFP with chromocentre heterochromatin and the fourth chromosome. In vivo confocal section of a salivary gland nucleus. (B) Relocation of SUV39H1–EGFP to euchromatic sites in transgenic lines with HP1/PC chimeric protein. (C and D) SUV39H1 rescues the dominant suppressor effect of Su(var)3–9 mutations: (C) control w^m4/Y; Su(var)3–9⁰⁶/+ and (D) w^m4/Y; Su(var)3–9⁰⁶/pP{GS[ry⁺, hs cDNA SUV39H1–EGFP]} males.