The DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. DHR3 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Here we provide evidence that DHR3 directly contributes to both of these regulatory responses. DHR3 protein is bound to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory genes, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Three DHR3 binding sites were identified downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of DHR3 regulation. Ectopic expression of DHR3 revealed that the polytene chromosome binding pattern is of functional significance. DHR3 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. DHR3 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae. This study also provides evidence for direct cross-regulation among orphan members of the nuclear receptor superfamily and further implicates these genes as critical transducers of the hormonal signal during the onset of Drosophila metamorphosis.

REFERENCES

REFERENCES
Ashburner
M.
(
1974
)
Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibitors of protein synthesis.
Dev. Biol
39
,
141
157
Ashburner
M.
,
Chihara
C.
,
Meltzer
P.
,
Richards
G.
(
1974
)
Temporal control of puffing activity in polytene chromosomes.
Cold Spring Harbor Symp. Quant. Biol
38
,
655
662
Ayer
S.
,
Walker
N.
,
Mosammaparast
M.
,
Nelson
J. P.
,
Shilo
B.
,
Benyajati
C.
(
1993
)
Activation and repression of Drosophila alcohol dehydrogenase distal transcription by two steroid hormone receptor superfamily members binding to a common response element.
Nucl. Acids Res
21
,
1619
1627
Baehrecke
E. H.
,
Thummel
C. S.
(
1995
)
The Drosophila E93 gene fromthe 93F early puff displays stage-and tissue-specific regulation by 20-hydroxyecdysone.
Dev. Biol
171
,
85
97
Boyd
L.
,
O'Toole
E.
,
Thummel
C. S.
(
1991
)
Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila.
Development
112
,
981
995
Burtis
K. C.
,
Thummel
C. S.
,
Jones
C. W.
,
Karim
F. D.
,
Hogness
D. S.
(
1990
)
The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets -related proteins.
Cell
61
,
85
99
Clever
U.
(
1964
)
Actinomycin and puromycin: effects on sequential gene activation by ecdysone.
Science
146
,
794
795
DiBello
P. R.
,
Withers
D. A.
,
Bayer
C. A.
,
Fristrom
J. W.
,
Guild
G. M.
(
1991
)
The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers.
Genetics
129
,
385
397
Emery
I. F.
,
Bedian
V.
,
Guild
G. M.
(
1994
)
Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis.
Development
120
,
3275
3287
Fletcher
J. C.
,
Burtis
K. B.
,
Hogness
D. S.
,
Thummel
C. S.
(
1995
)
The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone.
Development
121
,
1455
1465
Fletcher
J. C.
,
Thummel
C. S.
(
1995
)
The Drosophila E74 gene is required for the proper stage-and tissue-specific transcription of ecdysone-regulated genes at the onset of metamorphosis.
Development
121
,
1411
1421
Forman
B. M.
,
Chen
J.
,
Blumberg
B.
,
Kliewer
S. A.
,
Henshaw
R.
,
Ong
E. S.
,
Evans
R. M.
(
1994
)
Cross-talk among ROR1 and the Rev-erb family of orphan nuclear receptors.
Mol. Endo
8
,
1253
1261
Giguere
V.
,
McBroom
L. D. B.
,
Flock
G.
(
1995
)
Determinants of target gene specificity for ROR1: monomeric DNA binding by an orphan nuclear receptor.
Mol. Cell. Biol
15
,
2517
2526
Giguere
V.
,
Tini
M.
,
Flock
G.
,
Ong
E.
,
Evans
R. M.
,
Otulakowski
G.
(
1994
)
Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR, a novel family of orphan hormone nuclear receptors.
Genes Dev
8
,
538
553
Guay
P. S.
,
Guild
G. M.
(
1991
)
The ecdysone-induced puffing cascade in Drosophila salivary glands: a Broad-Complex early gene regulates intermolt and late gene transcription.
Genetics
129
,
169
175
Guo
L. H.
,
Stepien
P. P.
,
Tso
J. Y.
,
Brousseau
R.
,
Narang
S.
,
Thomas
D. Y.
,
Wu
R.
(
1984
)
Synthesis of human insulin gene VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli.
Gene
29
,
251
254
Harding
H. P.
,
Lazar
M. A.
(
1993
)
The orphan receptor Rev-ErbAactivates transcription via a novel response element.
Mol. Cell. Biol
13
,
3113
3121
Harding
H. P.
,
Lazar
M. A.
(
1995
)
The monomer-binding orphan receptor Rev-erb represses transcription as a dimer on a novel direct repeat.
Mol. Cell. Biol
15
,
4791
4802
Hope
I. A.
,
Struhl
K.
(
1985
)
GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast.
Cell
43
,
177
188
Horner
M.
,
Chen
T.
,
Thummel
C. S.
(
1995
)
Ecdysone regulation and DNA binding properties of Drosophila nuclear hormone receptor superfamily members.
Dev. Biol
168
,
490
502
Huet
F.
,
Ruiz
C.
,
Richards
G.
(
1993
)
Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila.
Development
118
,
613
627
Huet
F.
,
Ruiz
C.
,
Richards
G.
(
1995
)
Sequential gene activation by ecdysone in Drosophila melanogaster: the hierarchical equivalence of early and early late genes.
Development
121
,
1195
1204
Karim
F. D.
,
Guild
G. M.
,
Thummel
C. S.
(
1993
)
The DrosophilaBroad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis.
Development
118
,
977
988
Karim
F. D.
,
Thummel
C. S.
(
1991
)
Ecdysone coordinates the timing and amounts of E74A and E74B transcription in Drosophila.
Genes Dev
5
,
1067
1079
Karim
F. D.
,
Thummel
C. S.
(
1992
)
Temporal coordination of regulatory gene expression by the steroid hormone ecdysone.
EMBO J
11
,
4083
4093
Kiss
I.
,
Beaton
A. H.
,
Tardiff
J.
,
Fristrom
D.
,
Fristrom
J. W.
(
1988
)
Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster.
Genetics
118
,
247
259
Koelle
M. R.
,
Segraves
W. A.
,
Hogness
D. S.
(
1992
)
DHR3: a Drosophila steroid receptor homolog.
Proc. Natl. Acad. Sci. USA
89
,
6167
6171
Koelle
M. R.
,
Talbot
W. S.
,
Segraves
W. A.
,
Bender
M. T.
,
Cherbas
P.
,
Hogness
D. S.
(
1991
)
The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily.
Cell
67
,
59
77
Lavorgna
G.
,
Karim
F. D.
,
Thummel
C. S.
,
Wu
C.
(
1993
)
Potential role for a FTZ-F1 steroid receptor superfamily member in the control of Drosophila metamorphosis.
Proc. Natl. Acad. Sci. USA
90
,
3004
3008
Mangelsdorf
D. J.
,
Evans
R. M.
(
1995
)
The RXR heterodimers and orphan receptors.
Cell
83
,
841
850
Mangelsdorf
D. J.
,
Thummel
C.
,
Beato
M.
,
Herrlich
P.
,
Schutz
G.
,
Umesono
K.
,
Blumberg
B.
,
Kastner
P.
,
Mark
M.
,
Chambon
P.
,
Evans
R. M.
(
1995
)
The nuclear receptor superfamily: The second decade.
Cell
83
,
835
839
Maroni
G.
,
Stamey
S. C.
(
1983
)
Use of blue food to select synchoronous, late third instar larvae.
Dros. Inf. Serv
59
,
142
143
Ohno
C. K.
,
Petkovich
M.
(
1992
)
FTZ-F1, a novel member of the Drosophila nuclear receptor family.
Mech. Dev
40
,
13
24
Palli
S. R.
,
Hiruma
K.
,
Riddiford
L. M.
(
1992
)
An ecdysteroid-inducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily.
Dev. Biol
150
,
306
318
Reuter
R.
,
Scott
M. P.
(
1990
)
Expression and function of the homeotic genes Antennapedia and Sex combs reduced in the embryonic midgut of Drosophila.
Development
109
,
289
303
Richards
G.
(
1976
)
Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster: V. The late prepupal puffs.
Dev. Biol
54
,
264
275
Robertson
C. W.
(
1936
)
The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principle morphological changes.
J. Morphol
59
,
351
399
Rubin
G. M.
,
Spradling
A. C.
(
1982
)
Genetic transformation of Drosophila with transposable element vectors.
Science
218
,
348
353
Russell
S. R. H.
,
Heimbeck
G.
,
Goddard
C. M.
,
Carpenter
A. T. C.
,
Ashburner
M.
(
1996
)
The Drosophila Eip78C gene is not vital but has a role in regulating chromosome puffs.
Genetics
144
,
159
170
Segraves
W. A.
,
Hogness
D. S.
(
1990
)
The E75 ecdysone-inducible generesponsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily.
Genes Dev
4
,
204
219
Seol
W.
,
Choi
H.-S.
,
Moore
D. D.
(
1996
)
An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors.
Science
272
,
1336
1339
Sliter
T. J.
,
Gilbert
L. I.
(
1992
)
Developmental arrest and ecdysteroid deficiency resulting from mutations at the dre4 locus of Drosophila.
Genetics
130
,
555
568
Stone
B. L.
,
Thummel
C. S.
(
1993
)
The Drosophila 78C early late puff contains E78, an ecdysone-inducible gene that encodes a novel member of the nuclear hormone receptor superfamily.
Cell
75
,
307
320
Talbot
W. S.
,
Swyryd
E. A.
,
Hogness
D. S.
(
1993
)
Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms.
Cell
73
,
1323
1337
Thomas
H. E.
,
Stunnenberg
H. G.
,
Stewart
A. F.
(
1993
)
Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle.
Nature
362
,
471
475
Thummel
C. S.
(
1995
)
From embryogenesis to metamorphosis: The regulation and function of Drosophila nuclear receptor superfamily members.
Cell
83
,
871
877
Thummel
C. S.
(
1996
)
Flies on steroids: Drosophila metamorphosis and the mechanisms of steroid hormone action.
Trends Genet
12
,
306
310
Thummel
C. S.
,
Burtis
K. C.
,
Hogness
D. S.
(
1990
)
Spatial and temporal patterns of E74 transcription during Drosophila development.
Cell
61
,
101
111
Thummel
C. S.
,
Pirrotta
V.
(
1992
)
New pCaSpeR P element vectors.
Dros. Info. Serv
71
,
150
–.
Tini
M.
,
Fraser
R. A.
,
Giguere
V.
(
1995
)
Functional interactions between retinoic acid receptor-related orphan nuclear receptor (ROR) and the retinoic acid receptors in the regulation of the F-crystallin promoter.
J. Biol. Chem
270
,
20156
20161
Tsai
M.-J.
,
O'Malley
B. W.
(
1994
)
Molecular mechanisms of action of steroid/thyroid receptor superfamily members.
Annu. Rev. Biochem
63
,
451
486
Urness
L. D.
,
Thummel
C. S.
(
1990
)
Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein.
Cell
63
,
47
61
Weber
U.
,
Siegel
V.
,
Mlodzik
M.
(
1995
)
pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4.
EMBO J
14
,
6247
6257
Woodard
C. T.
,
Baehrecke
E. H.
,
Thummel
C. S.
(
1994
)
A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone.
Cell
79
,
607
615
Yao
T.
,
Forman
B. M.
,
Jiang
Z.
,
Cherbas
L.
,
Chen
J. D.
,
McKeown
M.
,
Cherbas
P.
,
Evans
R. M.
(
1993
)
Functional ecdysone receptor is the product of EcR and Ultraspiracle genes.
Nature
366
,
476
479
Yao
T.
,
Segraves
W. A.
,
Oro
A. E.
,
McKeown
M.
,
Evans
R. M.
(
1992
)
Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation.
Cell
71
,
63
72
Zink
B.
,
Paro
R.
(
1989
)
In vivo binding pattern of a transregulator of homeotic genes in Drosophila melanogaster.
Nature
337
,
468
471
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