Functional interactions between the Moses corepressor and DHR78 nuclear receptor regulate growth in Drosophila

Abstract

Expression of the Drosophila orphan nuclear receptor DHR78 is regulated by the steroid hormone ecdysone and is required for growth and viability during larval stages. In contrast to our understanding of its biological functions, however, relatively little is known about how DHR78 acts as a transcription factor. Here we show that DHR78 is an obligate partner for Moses (Middleman of seventy-eight signaling), a SAM (sterile alpha motif) domain-containing cofactor that requires DHR78 for its stability. Unlike other nuclear receptor cofactors, Moses has no obvious interaction domains and displays a unique binding specificity for DHR78. Moses acts as a corepressor, inhibiting DHR78 transcriptional activity independently of histone deacetylation. Consistent with their close association, DHR78 and Moses proteins are coexpressed during development and colocalize to specific genomic targets in chromatin. Moses mutants progress normally through early larval stages, like DHR78 mutants, but display an opposite overgrowth phenotype, with hypertrophy of adult tissues. Genetic interactions between DHR78 and moses result in a similar phenotype, suggesting that the relative dose of Moses and DHR78 regulates growth and prevents cancer. The tight functional association between DHR78 and Moses provides a new paradigm for understanding the molecular mechanisms by which cofactors modulate nuclear receptor signaling pathways.

Figure 1.

Identification of a specific DHR78-binding protein. (A) Schematic representation of yeast two-hybrid clones (D8 and D62), full-length version of D8 (78BP), D62A (amino acids 533–675), D62A1 (533–577), D62A2 (578–625), D62A3 (626–674), D62B (675–815), and two C-terminal truncations of 78BP, 78BPΔ1 (1–675) and 78BPΔ2 (1–533), with numbers representing amino acid positions. The DHR78 interaction domain (78ID) is pictured as a gray bar. (B) GST pulldowns show that GST-D8 and GST-D62, but not GST alone, bind to [³⁵S]Met-labeled DHR78 and show no interaction with DHR96, EcR, or USP. (C–F) Mammalian two-hybrid interaction assays in HEK293 cells. (C) GAL or GAL-DHR78 were tested for their ability to interact with a VP16 control, VP16–78BP, VP16–D62, and VP16–D62 constructs as depicted. (D) VP16–DHR78 interaction with GAL-D62A1 but not GAL-D62A2 or GAL-D62A3 defines the 78ID between amino acids 533 and 577. (E) The C-terminally truncated GAL-DHR78ΔAF2 fails to associate with VP16–78BP. (F) A survey of mammalian and Drosophila GAL-LBD fusions shows that VP16–78BP interacts significantly with only DHR78 and TR2.

Figure 2.

The DHR78-binding protein acts as a corepressor. (A, lanes 1–4) Electrophoretic mobility shift assay showing that in the absence of DHR78, increasing amounts of 78BP fail to associate with a ³²P-labeled CRPBII element. In contrast, DHR78 alone binds to the CRBPII element (lane 5), and this complex does not migrate into the gel upon the addition of increasing amounts of 78BP (lanes 6–8). Microliters of in vitro translated protein are shown. (B) VP16–DHR78 activates a tk-CRBPII-luc reporter gene and this response is repressed upon adding increasing amounts of a D8 expression plasmid (CMX-D8). (C) Cotransfection of HEK293 cells showing that GAL-fused constructs of 78BP mediate transcriptional repression through at least two separate domains, D62B and D62A2. (D) GAL-fused constructs of 78BP mediate transcriptional repression both in the absence (open bars) and presence (black bars) of 1 μM TSA in HEK293 cells.

Figure 3.

Moses binds to itself and DHR78, and acts as a transcriptional repressor. (A) Schematic representation of full-length Moses (amino acids 1–965), C-terminally truncated Moses (78BPΔ3, amino acids 1–791), the Moses SAM domain (amino acids 792–859), an N-terminal Moses deletion (C-term, amino acids 851–965), and the SAM domain deletion (ΔSAM, deletion of amino acids 806–851). The hatched box on 78BP (amino acids 835–885) represents where its sequence differs from that of Moses. The DHR78 interaction domain (78ID) is pictured as a gray bar. (B) GST pulldowns showing that GST-78BPΔ3, GST-ΔSAM, and GST-Moses, but not GST, GST-SAM, or GST-C-term, bind to [³⁵S]Met-labeled DHR78, Moses, and ΔSAM. (C) GAL-Moses and GAL-ΔSAM inhibit basal transcription over GAL alone, in the absence (open bars) or presence (black bars) of 1 μM TSA in HEK293 cells.

Figure 4.

Spatial and temporal expression profiles of Moses and DHR78. (A) Northern blot analysis to detect moses and DHR78 transcripts of w¹¹¹⁸ animals staged as mid-L3 (−18 and −8 h), late L3 (−4 h), newly formed prepupae (0 h), prepupae staged at 2-h intervals (2, 4, 6, 8, and 10 h), and pupae (12 and 14 h). Hours are relative to puparium formation. Hybridization to detect rp49 mRNA was included as a control for loading and transfer. (B–Q) Fat body and trachea from either −18-h mid-L3 or 0-h prepupae were stained with DAPI (B–E), DHR78 antibodies (F–I), or Moses antibodies (J–M). (N–Q) A merge of the antibody patterns. White arrows mark nuclei that contain DHR78 and Moses protein. (R–T) Antibody stains of giant salivary gland polytene chromosomes from late L3 (−4 h) w¹¹¹⁸ animals (wt) to detect DHR78 (R), Moses (S), and the merge of the two (T) bound to chromatin. (U) Antibody stains of Moses in hs-DHR78/hs-DHR78; DHR78²/DHR78² mutants that have been rescued to late L3 (−4 h) by transient expression of a DHR78 transgene during embryogenesis for 30 min at 37°C and maintained at 18°C through development, showing loss of chromatin localization.

Figure 5.

Moses protein is stabilized by DHR78. Fat bodies stained with DAPI (A–E), or antibodies directed against DHR78 (F–J) or Moses (K–O), from either mid-L3 (A–D,F–I,K–N) or 0-h prepupae (E,J,O). Either hs-DHR78 transformants, hs-moses transformants, or DHR78² mutants were examined, as indicated. The DHR78² mutants were rescued to later stages as described in the legend for Figure 4U. Animals were heat treated (+) or not (−) for 1 h at 37°C and then allowed to develop for 3 h prior to collection. (P) Northern blot analysis of w¹¹¹⁸, hs-DHR78, hs-moses animals, and DHR78² mutants, staged and treated as described above, showing that ectopic expression of DHR78 and moses, and the DHR78² point mutation, do not significantly affect the levels of endogenous moses and DHR78 mRNA. Hybridization to detect rp49 mRNA was included as a control. (Q) Western blot analysis of protein extracts from heat-treated w¹¹¹⁸ and the hs-moses transformant at the indicated times relative to pupariation, using antibodies against Moses, showing that Moses is unstable at a time when DHR78 protein is low (−18 h), but not when DHR78 is high (0 h). The relative level of protein assayed is 10-fold higher for w¹¹¹⁸ lanes (equivalent to 0.5 animal per lane) than hs-moses lanes (equivalent to 0.05 animal per lane).

Figure 6.

Mutations in moses are lethal and lead to uncontrolled growth. (A) Schematic representation of the moses locus on chromosome 2 shown from 5′ (left) to 3′ (right), with UTRs (white boxes) and protein-coding regions (black boxes) as well as the sites of two P-element insertions used for imprecise excisions. (B) Graph showing the viability of staged moses^5D/moses²⁴ animals relative to internal control heterozygotes from a total of 800 embryos that were followed through development. The lethality of moses^5D/moses²⁴ mutants can be rescued to adulthood with increasing levels of moses (hs-moses/+ and hs-moses/hs-moses, n = 400 for each). (C) The phenotypes of w¹¹¹⁸ control or moses^5D/moses²⁴ mutants are shown as late L3 (left two panels) or early pupae (right two panels). moses mutants pictured are 7–10 d older than the controls in order to show the overgrowth phenotype. All moses mutants that arrest as late L3 or prepupae display overgrowth and disc hyperplasia. (D) Heat-treated moses²⁴/+;hs-DHR78/+ L3 (right panel), but not hs-DHR78/+ control L3 (left panel), display an overgrowth phenotype indistinguishable from moses^5D/moses²⁴ mutants. (E) Late L3 brains and wing imaginal discs from control w¹¹¹⁸ or moses^5D/moses²⁴ mutants. Note the fused haltere (black arrow) and leg (white arrow) imaginal discs in mutants.

Figure 7.

Moses levels affect GAL4-DHR78 activity and stability. Trachea (A–C), salivary glands (D–F), and fat bodies (G–I) from hs-GAL4-DHR78/UAS-nlacZ late L3 that are either wild type for moses (+/+), or with one (+/moses^5D) or two (moses^5D/moses²⁴) mutant copies of moses. Animals were heat treated at 37°C for 30 min to induce GAL4-DHR78 expression prior to staging and collection. Nuclear β-galactosidase was detected by X-gal staining. Fat bodies from animals described above were stained with DAPI (J–L) or anti-GAL4 antibodies to detect the GAL4-DHR78 fusion protein (M–O). Similar results to those shown for +/moses^5D were seen in +/moses²⁴ animals (data not shown).

Figure 8.

A model for functional interactions between DHR78 and Moses. Either endogenous DHR78 (left half) or heat-induced GAL4-DHR78 fusion protein (right half) are depicted, along with different doses of Moses protein. Wild-type (A,D), moses/+ (B,E), or moses/moses mutant (C,F) genetic backgrounds are shown. The curved arrow represents the degradation of unstable protein. See text for a detailed discussion of the model.