The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development - PubMed (original) (raw)

The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development

Marianne Stabell et al. Nucleic Acids Res. 2006.

Abstract

Mammalian G9a is a histone H3 Lys-9 (H3-K9) methyltransferase localized in euchromatin and acts as a co-regulator for specific transcription factors. G9a is required for proper development in mammals as g9a-/g9a- mice show growth retardation and early lethality. Here we describe the cloning, the biochemical and genetical analyses of the Drosophila homolog dG9a. We show that dG9a shares the structural organization of mammalian G9a, and that it is a multi-catalytic histone methyltransferase with specificity not only for lysines 9 and 27 on H3 but also for H4. Surprisingly, it is not the H4-K20 residue that is the target for this methylation. Spatiotemporal expression analyses reveal that dG9a is abundantly expressed in the gonads of both sexes, with no detectable expression in gonadectomized adults. In addition we find a low but clearly observable level of dG9a transcript in developing embryos, larvae and pupae. Genetic and RNAi experiments reveal that dG9a is involved in ecdysone regulatory pathways.

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Figures

Figure 1

Figure 1

The domain organization is conserved between dG9a and G9a. (A) Alignment of SET domains and flanking cysteine-rich regions of mouse and Drosophila dG9a protein. The degree of conservation is distinguished at four levels (100, 80 and 60%, and not conserved), where 100% has the darkest shade of grey. The upper and lower case letters in the consensus line indicate 100 and 80% conservation within all groups, respectively. Numbers in the consensus line represent conserved similarity groups as defined by the Blossum 62 scoring table. The conserved R(H)ΦΦNHSC and the FDYG motifs are underlined. (B) Domain organization within Drosophila protein dG9a. An AT-hook and an ankyrin motif are found in addition to the SET domain.

Figure 2

Figure 2

The dG9a protein localizes to distinct chromosome bands. (A) Antibodies against dG9a raised in rabbit are specific and recognize a single band of ∼180 kDa as predicted on a western blot of Drosophila nuclear extract. (B) dG9a protein (in red) localizes to chromatin and gives a distinct banding pattern on polytene chromosomes. There is no staining in the chromocenter (arrow), and dG9a localizes predominantly to euchromatic regions. DNA is counterstained with DAPI (in blue).

Figure 3

Figure 3

Spatiotemporal expression of dG9a. (A) Developmental RT–PCR shows that dG9a is maternally deposited in the egg, and that there is moderate expression during the larval development. dG9a is present in all developmental stages investigated. (B–K) dG9a is present from the very start of oogenesis through the end of oogenesis in wild-type ovarioles. Anterior is to the left, posterior to the right. dG9a in red (right column) and the nuclei is counter stained with DAPI in blue (left column). (B and C) The early stages of oogenesis development. The dG9a protein is present from the very start. (D and E) Stage 10B ovaries. dG9a localizes to nuclei in both nurse and follicle cells. An accumulation of protein is observed in the region where the anterior polar cells and the centripetal follicle cells are located, arrowheads and in the posterior follicle cells, arrow. (F and G) Stage 11. Shortly after centripetal migration (stage 10B), the nurse cells rapidly transfer their contents into the oocyte (stage 11) then begin to degenerate and undergo apoptosis (stages 12–14). (H and I) Stage 12. Dumping complete, no or very little dG9a is detectable in the degenerating nurse cell nuclei, but is still present in the follicle cells. Notice the accumulation of dG9a protein in the extreme posterior part of the egg, arrowheads, where the posterior polar cells located. (J and K) Stage 14. The egg is fully developed and dG9a protein is maternally deposited. (L–S) Lateral views of wild-type embryos hybridized with digoxigenin-labeled RNA probes (L and N with Nomarski optics) or with a dG9a antibody (M, O, Q and S). Anterior is to the left and dorsal is up. The nuclei are counter stained with DAPI in blue. (L and M) Embryo at syncytial blastoderm stage (stage 4, ∼1.5–2.5 h). dG9a is localized to the nuclei. In early embryos the message and the protein are ubiquitously distributed due to its maternal contribution. (N and O) Embryo during germband extension (stage 9). (P and Q) Stage 12. In late-stage embryos, expression is strongest in the CNS and the neuroectoderm. (K and S) Stage 13. Surface view of embryo at the completion of germband shortening.

Figure 4

Figure 4

Characterization of recombinant dG9a. (A) Eluted FLAG tagged dG9a (789–1637 amino acid) was separated on a 12% SDS–PAGE and stained with Coomassie blue G250. (B) In vitro methylation reactions using dG9a (lanes 1–6), no enzyme (lane 7) and dSu(var)3–9 (lane 8). In the reaction we used 1 µg of different histones: recombinant histone H3 (lane 1), recombinant histone H4 (lane 2), recombinant (lane 3) and native histone octamer (lane 5) and recombinant and native nucleosomes (lanes 4 and 6) reconstituted on circular pBS(KS) from equimolar amounts of histones. The upper panel shows Coomassie stained gel and the lower panel the autoradiograph. (C) Activity of recombinant mouse G9a expressed in baculovirus infected cells (a kind gift from S. Pradhan). HKMTase activity on 1 µg of different histone substrates: recombinant histone H3 (lane 1), recombinant histone H4 (lane 2), recombinant and native histone octamers (lanes 3 and 5) and recombinant and native nucleosomes (lanes 4 and 6). Mock control (lane 7) is incubation of recombinant octamer without enzyme. The Coomassie gel is shown at the top and the corresponding autoradiograph at the bottom. (D) FLAG dG9a wild type versus H1536K mutation of the conserved region of the SET domain. The upper panels shows a western blot of the two proteins. Recombinant octamer (2 µg) was used as substrate for 25. 50 and 100 ng of wt (lanes 1–3) and H1536K mutant (lanes 4–6). The corresponding autoradiograph is shown in the lower panel. (E) In vitro methylation of 2 µg of recombinant H3 (lane 1), H3 mutated at lysine 9 (lane 2), H3 mutated at lysine 27 (lane 3) or both (lane 4) using dG9a and a mock purification. Coomassie stained H3 is shown in the upper panel and a corresponding autoradiography in lower panel. A corresponding filter binding assay is shown to the right. The _y_-axis displays the percent radioactivity incorporated on 2 µg of histone H3 and H3 mutants K9A, K27A and K9/K27A with radioactivity incorporated on wt H3 set to 100% and the background is subtracted. (F) HKMTase activity of mG9a on histone H3 molecules and H3 K9A, H3 K27A and the double mutant K9A/K27A. A gel of Coomassie stained histones and the corresponding autoradiography is shown. On the right, a filter binding assay showing percent radioactivity incorporated on 2 µg of histone H3 and H3 mutants K9A, K27A and K9/K27A. The _y_-axis displays the percent radioactivity incorporated with activity on wt H3 set to 100% and the background is subtracted (G) Amino acid sequence of the H4 N-terminus is shown at the top. Asterisks indicate possible substrates for dG9a in vitro. Methylation of 2 µg of recombinant H4 (lane 1), H4 K20A (lane 2), H4Δ5 (lane 3), H4Δ10 (lane 4), H4Δ15 (lane 5) and globular H4 (lane 6). Mock control (lane 7) is incubation of wt H4 without enzyme. (H) Quantitative MALDI-TOF analysis of 500 ng of H3 and H4 methylated by 100 ng of dG9a. Peptides spanning amino acids 9–17 and 27–40 of H3 and 4–17 of H4 is represented by graphs. No signals were observed in other peptides. Mono-, di- and trimethylation are shown as percent of total H3 or H4. This figure is representative for at least three different methyltranferase assays.

Figure 5

Figure 5

Knock down of dG9a give phenotypic effects. RNAi experiments show that dG9a is important for development. (A) RT–PCR shows that dG9a is down regulated by RNAi; rp49 is used as loading control. (B and C) Using a ubiquitously expressed driver, da-GAL4, to induce the IR construct shows that dG9a is required for proper transition from larva to pupa. Penetrance is 100%. In (B) the larva is 6 days, in (C) 8 days. The IR construct is tagged with an independent UAS-GFP, which can be used as control (insert). Melanotic tumors are frequently observed in these larvae. (D and E) Using a larval CNS-GAL4 (BL 3739) driver the progeny makes it up to and through pupariation (F), but fails to hatch. Differentiation seems more complete in posterior part of the animal (G). A similar phenotype is observed when using Act5C-GAL4 as driver. Remarkably, in both cases, the escapers observed to hatch (∼10%) are females. (H and I) When using the ap-GAL4 driver defects in the wings are observed. This phenotype is highly pleiotropic, with one or both wings affected. Among the phenotypes are narrow wings held in a Dichaeate-like fashion, wings standing straight up and blistered wings. Progeny with no apparent defects are also observed. (J) Progeny of genotype ap-GAL4,UAS-dG9a.IR/EcRM544fs show a wing phenotype of severe character and 100% penetrance.

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