Hormone-receptor-like in 78


Targets of Activity

In a search for retinoid X receptor-like molecules in Drosophila, an additional member of the nuclearreceptor superfamily, HR78 or XR78E/F, has been identified. HR78 binds as a homodimer to directrepeats (DR1 spaced by 1 nucleotide) of the sequence AGGTCA. In transient transfection assays, HR78 represses ecdysone signaling in aDNA-binding-dependent fashion. HR78 has its highest expression in third-instar larvae and prepupae. Hr78 inhibits ecdysone signaling on the Eip28/29 Ecdysone response element but not the hs27 Ecdysone response element. Perhaps Hr78 competes for Ecdysone receptor/Ultraspiracle on one element and not the other. A DR3 is present on Eip28/29 and not on hs27. These results suggest that Hr78 inhibits ecdysone responsiveness from the Eip28/29 element by direct competition for binding to the regulatroy DNA sites and that Hr78 fails to inhibit ecdysone responseness on the hs27 EcRE because it cannot bind to this element. Theseexperiments suggest that HR78 may play a regulatory role in the transcriptional cascade triggered by the hormoneecdysone in Drosophila (Zelhof, 1995).

Three Drosophila genes, designated HR38, HR78, and HR96, have been isolated. All three genes are expressed throughout third-instar larvaland prepupal development. HR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B responseelement. HR78 and HR96 are orphan receptor genes. HR78 is induced by 20-hydroxyecdysone (20E) in culturedlarval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats.HR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20Ereceptor can bind to each of the sequences recognized by HR78 and HR96, indicating that these proteins may competewith the receptor for binding to a common set of target sequences (Fisk, 1995).

A number of chromosomal aberrations have been generated that disrupt the early-late ecdysone-induced 78C puff gene(Eip78C or Hr78), which encodes the twomembers of the nuclear hormone receptor superfamily Hr78-A and Hr78-B. The aberrations include deletions of theligand-binding/dimerization domain of both, inversions that split Hr78-A but retain residual Hr78-B expression, and asmall deletion specific for Hr78-B. Wild-type Hr78 functions are completely dispensable for normaldevelopment under laboratory conditions. However, Hr78-B is required for the maximal puffing activity ofa subset of late puffs (63E and 82F) since these puffs are reduced in size in Hr78-B mutant backgrounds. Paradoxically,the same late puffs are reduced, as well as at least one other, when the Hr78-B cDNA is overexpressed from a heat shockpromoter. These data indicate either that Hr78 function is redundant or that it plays a subtle modulating role in theregulation of chromosome puffing (Russell, 1996).

The expression of Hr78 in larval salivary glands allowed for the identification of potential regulatory targets by antibody staining ofpolytene chromosomes. Whereas no binding sites can be detected in polytene chromosomes prepared from Hr78 mutant larvae, multiple stained sites could be detected in polytene chromosomes prepared from wild-type mid-third instar larvae. Hr78 protein can bind to a subset of 20E receptor binding sites in vitro, suggesting that Hr78 might function at the top of the ecdysteroid regulatory hierarchies. In order to determine if Hr78 exhibits a similar binding specificity in vivo, polytene chromosomes were stained with antibodies directed against either Hr78 or Ultraspiracle. The staining pattern of Usp is identical to that of EcR, and thus indicative of sites bound by the 20E receptor. Some sites are bound primarily by the EcR or Hr78, while the majority of sites are bound by both proteins, consistent with an overlap in their binding specificity. In order to map the sites bound by Hr78, salivary glands were dissected from newly formed white prepupae, when Hr78 protein is most abundant, and polytene chromosome preparations were stained with anti-Hr78 antibodies. Over 100 Hr78 binding sites have been identified, many of which correspond to ecdysteroid regulated puff loci (Fisk, 1998).

Protein Interactions and Post-transcriptional regulation

To verify that the phenotypes observed are caused by mutations in Hr78, an attempt was made to rescue the mutants by ectopically expressing Hr78 from the heat-inducible hsp70 promoter. Western analysis of extracts prepared from heat-treated transformants demonstrates that these animals express approximately 10-fold higher levels of Hr78 protein than is normally present in late third instar larvae. Daily treatments at 37°C for 30 min result in an efficient rescue of the pupariation defect. Furthermore, at least 95% of the mutant animals that pupariated proceed to eclose as normal, fertile adults. Ectopic expression of Hr78 during embryogenesis and first instar larval development is less efficient at rescuing puparium formation than later heat pulses, suggesting that Hr78 function is required during the later stages of larval development. Interestingly, heat-treated wild-type animals, either with or without the hsp70-Hr78 transgene, showed no detectable phenotypes, indicating that Hr78 activity is regulated posttranslationally (Fisk, 1998).

Hormone-receptor-like in 78: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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