DNA binding and transactivation characteristics of the mosquito ecdysone receptor-Ultraspiracle complex.
The steroid hormone 20-hydroxyecdysone is a key regulatory factor, controlling blood-meal triggered egg maturation in mosquitoes. To elucidate the ecdysone hierarchy governing this event, we cloned and characterized the ecdysone receptor (AaEcR) and the nuclear receptor Ultraspiracle (AaUSP), a retinoid X receptor homologue, from the mosquito, Aedes aegypti, which form a functional complex capable of ligand and DNA binding. Here we analyzed the DNA-binding properties of the AaEcR.AaUSP heterodimer with respect to the effects of nucleotide sequence, orientation, and spacing between half-sites in natural Drosophila and synthetic ecdysone response element (EcREs). By using an electrophoretic gel mobility shift assay, we showed that AaEcR.AaUSP exhibits a broad binding specificity, forming complexes with inverted (IR) and direct (DR) repeats of the nuclear receptor response element half-site consensus sequence AGGTCA separated by spacers of variable length. A single nucleotide spacer was optimal for both imperfect (IRhsp-1) and perfect (IRper-1) inverted repeats; adding or removing 1 base pair in an IRhsp-1 spacer practically abolished binding. However, changing the half-site to the consensus sequence AGGTCA (IRper-1) increased binding of AaEcR.AaUSP 10-fold over IRhsp-1 and, at the same time, reduced the stringency of the spacer length requirement, with IRper-0 to IRper-5 showing detectable binding. Spacer length was less important in DRs of AGGTCA (DR-0 to DR-5); although 4 bp was optimal, DR-3 and DR-5 bound AaEcR.AaUSP almost as efficiently as DR-4. Furthermore, AaEcR. AaUSP also bound DRs separated by 11-13 nucleotide spacers. Competition experiments and direct estimation of binding affinity (Kd) indicated that, given identical consensus half-sites and an optimal spacer, the AaEcR.AaUSP heterodimer bound an IR with higher affinity than a DR. Co-transfection assays utilizing CV-1 cells demonstrated that the mosquito EcR.USP heterodimer is capable of transactivating reporter constructs containing either IR-1 or DR-4. The levels of transactivation are correlated with the respective binding affinities of the response elements (IRper-1 > DR-4 > IRhsp-1). Taken together, these analyses predict broad variability in the EcREs of mosquito ecdysone-responsive genes.
Wang
S F
SF
Programs in Genetics, Cell & Molecular Biology, Departments of Entomology, Michigan State University, East Lansing, Michigan 48824, USA.
Miura
K
K
Miksicek
R J
RJ
Segraves
W A
WA
Raikhel
A S
AS
D000330:Aedes / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins; D004440:Ecdysone / Q000378:metabolism; D017930:Genes, Reporter; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D011987:Receptors, Steroid / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009838:Oligodeoxyribonucleotides; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; D004440:Ecdysone