Atrophin recruits HDAC1/2 and G9a to modify histone H3K9 and to determine cell fates

Abstract

Atrophin family proteins, including the vertebrate arginine-glutamic acid dipeptide repeats protein (RERE) and Drosophila Atrophin (Atro), constitute a new class of nuclear receptor corepressors. Both RERE and Atro share the ELM2 (EGL-27 and MTA1 homology 2) and SANT (SWI3/ADA2/N-CoR/TFIII-B) domains, which are also present in other important transcriptional cofactors. Here, we report that the SANT domain in RERE binds to the histone methyltransferase G9a, and that both the ELM2 and SANT domains orchestrate molecular events that lead to a stable methylation of histone H3-lysine 9. We establish the physiological relevance of these interactions among Atrophin, G9a, and histone deacetylases 1 and 2 in Drosophila by showing that these proteins localize to overlapping chromosomal loci, and act together to suppress wing vein and melanotic-mass formation. This study not only shows a new function of the SANT domain and establishes its connection with the ELM2 domain, but also implies that a similar strategy is used by other ELM2-SANT proteins to repress gene transcription and to exert biological effects.

Figure 1

Arginine–glutamic acid dipeptide repeats protein exerts histone methylation activity and associates with G9a. (A) Diagram showing the Flag-tagged Atrophin (Atro) proteins used in immunoprecipitation experiments. The conserved BAH (bromo-adjacent homology), ELM2 (EGL-27 and MTA1 homology 2) and SANT (SWI3/ADA2/N-CoR/TFIII-B) domains in each protein are highlighted in different colours. (B) Histone methylation (HMT) assays were performed on recombinant histone octamers to determine whether the ELM2–SANT domains of arginine–glutamic acid dipeptide repeats protein (RERE) mediate HMT activity. The identity of each histone is determined by its size in the Coomassie blue-stained gel; G9a, a known histone H3-specific histone methyltransferase (HMTase), was used as a control. (C,D) HMT assays were performed on the indicated peptides to determine which specific lysine residue is a target of the RERE^ELSA complex. Recombinant SET9, a known H3K4 HMTase, was used as a control. (E) Western blot experiments were performed on the indicated immunoprecipitation products to test whether G9a is associated with Atro proteins. SET9 was used as a negative control. A cleaved product of RERE is marked with an asterisk. (F) Immunostaining experiments were performed on cells expressing green fluorescent protein (GFP)-tagged RERE or cyan fluorescent protein (CFP)-tagged Atro to determine whether endogenous G9a is recruited to the RERE/Atro-mediated nuclear foci. Nuclear localization signal-tagged CFP (NCFP) was used as a control. ELSA, ELM2+SANT domains; F, Flag; H3K9met2, dimethylated H3K9; IB, immunoblot; IP, immunoprecipitation; NF, nuclear localization signal-tagged Flag; WCE, whole-cell extract.

Figure 2

The SANT domain of arginine–glutamic acid dipeptide repeats protein binds to G9a and coordinates with the ELM2 domain to methylate H3K9. (A) Western blot experiments were performed on the indicated immunoprecipitation products to determine which region of arginine–glutamic acid dipeptide repeats protein (RERE) is responsible for G9a association. The input proteins are shown in the right panels. Cleaved products of RERE are marked with asterisks. Based on the molecular weights of the shown cleaved products of RERE, they all contain both ELM2 and SANT domains. (B) Glutathione-S-transferase (GST) pull-down experiments were used to determine whether the SANT domain of RERE binds to G9a directly. G9a and SET9 were labelled with [³⁵S]methionine. The expression of GST-RERE proteins used in the pull-down assays is shown by Coomassie blue staining. (C) Western blot experiments were performed on the indicated immunoprecipitation products to test whether G9a is associated with additional ELM2–SANT domain proteins. Histone deacetylases 1 and 2 (HDAC1/2) were used as positive controls. (D) Histone methyltransferase assays were performed on an acetylated form of H3(1–21)K9 to determine whether RERE^ELSA-mediated histone methylation is sensitive to trichostatin A (TSA) treatment. TSA is an HDAC inhibitor. ELM2, EGL-27 and MTA1 homology 2; ELSA, ELM2+SANT domains; G, GST; G9a, a known histone H3-specific histone methyltransferase; IB, immunoblot; IP, immunoprecipitation; MIER1, mesoderm induction early response 1; MTA1–3, metastasis-associated family proteins 1–3; NF, nuclear localization signal-tagged Flag; SANT, SWI3/ADA2/N-CoR/TFIII-B; SET9, an H3-K4 histone methyltransferase; WCE, whole-cell extract.

Figure 3

Atro, dG9a and Rpd3 bind to overlapping chromosomal regions in Drosophila. A co-immunostaining experiment was performed on salivary gland cells to determine the relative positions of Atro and dG9a, Rpd3 or a marker for transcriptional initiation (RNA polymerase II-phosphorylated-Ser5 (RNAP-p-S5)) on polytene chromosomes. Salivary gland cells derived from Hsp70∷Atro late third instar larvae were subjected to co-immunostaining using (A) Atro and dG9a antibodies, (B) Atro and Rpd3 antibodies, and (C) Atro and RNAP-p-S5 antibodies. Hsp70-Gal4 is a salivary gland-specific Gal4 driver without heat-shock treatment. The enlarged images shown in the right panels of (A) correspond to the boxed area in the lower panel. Atro, Drosophila Atrophin; dG9a, the Drosophila homologue of G9a; Hsp70, Heat-shock protein 70; Rpd3, the fly homologue of histone deacetylases 1 and 2.

Figure 4

Atro, dG9a and Rpd3 act together to suppress melanotic-mass formation in the adult head of Drosophila. (A) The expression of RedStinger was used to determine that dpp-Gal4 is active in the adult head. The images show posterior views of the heads of dpp-Gal4 and dpp∷RedStinger flies. RedStinger is a DsRed variant. (B) Examination of adult heads to determine whether Atro interacts genetically with dG9a and Rpd3. Images show posterior views of the heads from flies with the indicated genotypes. dpp∷Atro.IR1 is an Atro double-stranded RNA-expressing line, w¹¹¹⁸ and dpp∷GFP were used as negative control lines, dG9a^RG5 is a deletion line generated by homologous recombination and dG9a^Del34 is an imprecise P-element excision line. The bottom panel contains enlarged images corresponding to the boxed areas shown in the middle panel. The melanotic masses are indicated by asterisks. (C) Genetic data showing the number and percentage of flies with melanotic masses in their heads. The genotype of each examined fly line is indicated. Atro³⁵ is a null allele, Rpd3⁰⁴⁵⁵⁶ is a loss-of-function allele and Rpd3³¹³ is a hypomorphic allele. Atro, Drosophila Atrophin; dG9a, the Drosophila homologue of G9a; e, eye; ob, occipital bristles; of, occipital foramen; Rpd3, the fly homologue of histone deacetylases 1 and 2.