Differential control of MHR3 promoter activity by isoforms of the ecdysone receptor and inhibitory effects of E75A and MHR3.
MHR3 is an ecdysone-inducible transcription factor whose expression in both Manduca sexta epidermis and the Manduca GV1 cell line is induced by 20-hydroxyecdysone (20E) in vitro. There are four putative ecdysone response elements (EcRE) in the 2.6-kb flanking region of the MHR3 promoter. The most proximal, EcRE1, is necessary for activation of the promoter by 20E in the GV1 cells because the mutation of EcRE1 caused the loss of responsiveness to 20E. Previous studies showed that EcR-B1/USP-1 bound only to EcRE1 and high levels of this complex increased the 20E-induced activation, whereas the presence of high USP-2 prevented this increased activation. When we expressed EcR-A alone or in combination with USP-1 under the control of Autographa californica baculovirus promoter (pIE1hr), the activation of the 2.6-kb promoter by 20E was reduced by about 50%. Moreover, when EcR-A was expressed together with both EcR-B1 and USP-1, it reduced the normal activation caused by EcR-B1 and USP-1 by 50%. Gel mobility shift assays showed no binding of EcR-A/USP-1 to EcRE1. The presence of EcR-A, however, reduced the binding of EcR-B1/USP-1 by about 50%. These findings suggest that EcR-A competes with EcR-B1 for binding of USP-1, leading to a decline in activity of the promoter. In addition, E75A, another ecdysone-induced transcription factor, and MHR3 itself suppressed MHR3 promoter activity by binding to the monomeric response element (MRE2). Therefore, MHR3 can be down-regulated both by itself and by E75A.
Hiruma
Kiyoshi
K
Department of Biology, University of Washington, Seattle, WA 98195-1800, USA.
Riddiford
Lynn M
LM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics; D029721:Drosophila Proteins; D004441:Ecdysterone / Q000494:pharmacology; D004817:Epidermis / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D006023:Glycoproteins / Q000187:drug effects / Q000235:genetics; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism*; D018402:Manduca / Q000166:cytology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020033:Protein Isoforms; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D015843:Serpins; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006023:Glycoproteins; D019476:Insect Proteins; D020033:Protein Isoforms; D011987:Receptors, Steroid; D015843:Serpins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; C467903:ecdysone receptor A, insect; C110602:transcription factor E75, Manduca; C029708:uterine luminal fluid proteins; D004441:Ecdysterone