In this study, we identified a new cis-acting DNA regulatory element that confers JH-dependent gene expression. A JHRE, DmJHRE1, shared by 13 genes in the fruit fly and 12 genes in the honeybee of the 16 genes induced by JH in both D. melanogaster L57 cells and A. mellifera brain was identified. The DmJHRE1 was used in a DNA affinity column; two DmJHRE1-binding proteins, 39-kDa FK506-binding protein (FKBP39) and 21-kDa calponin-like protein (Chd64), were identified in Drosophila by LC-MS/MS; and the homologs were also found in A. mellifera.
Previously, Kethidi et al.
) identified a 30-base pair sequence (CfJHRE), located in the 5′ promoter region of the spruce budworm JH esterase gene (from −604 to −574) that showed 100% similarity with the consensus DR4 element. The CfJHRE is sufficient to support both JH-dependent induction and 20E suppression of JH induction. In L. migratoria
, a putative JHRE with the partially palindromic 15-nt sequence, GAGGTTCGAG(A/T)CCT(T/C), was found upstream of a JH-induced gene, jhp21
, and a transcription factor binding to this element was identified. The binding of a factor to the JHRE was abolished by phosphorylation catalyzed by protein kinase C and was brought to an active state through the action of JH (38
). The DmJHRE1 we identified in this study does not show sequence similarity with either of the previously identified JHREs, suggesting that JH may function through diverse response elements.
The hypothesis that guided this work is that given that JH coordinates the regulation of many essential response genes, some of these co-regulated genes may share common response elements and regulatory factors (49
). Therefore, JHRE sequences may be evolutionarily conserved in D. melanogaster
and A. mellifera
. Sinha et al.
) reported on evolutionary conservation of cis
-regulatory elements between D. melanogaster
and A. mellifera
. Identification of cis
-regulatory elements is helpful to investigate the molecular mechanism of JH action, because cis
-regulatory elements could be involved in both temporal and spatial regulation of genes regulated by JH. In this study, we used the MEME algorithm and identified a JHRE, DmJHRE1, shared by 13 of the 16 JH-induced genes identified in D. melanogaster
L57 cells and also shared by 12 of the 16 JH-induced genes identified in A. mellifera
Transient transfection experiments in D. melanogaster
L57 cells were used to confirm the hypothesis that DmJHRE1 supports induction of genes placed under its control. DmJHRE1 is also present in other JH-induced genes in Drosophila
(a list of JH-induced genes is given in Ref. 40
). We extended the analysis to more evolutionarily divergent insect species, whose genomes have been sequenced and are available in public data bases. DmJHRE1 is present in the promoters of some genes in A. mellifer
and Anopheles gambiae
, indicating that DmJHRE1 is conserved during evolution.
Two DmJHRE1-binding proteins, 21-kDa calponin-like protein (Chd64) and 39-kDa FK506-binding protein (FKBP39), were identified. Previous RNAi experiments showed that FKBP39
is involved in Wg signaling (51
). The homolog of FKBP39 in fall armyworm Spodoptera frugiperda
was identified as a nuclear DNA-binding protein associated with a nuclear kinase that specifically phosphorylates it in the presence of Mg2+
and ATP (52
). Nuclear FKBPs could help to maintain kinase function by maintaining the proper protein-protein interactions in the kinase complex, and this complex in turn might play a role in signal transduction by aiding in proper folding and stability of short lived labile transcription factors (52
). The FKB39 homologs were also found in A. mellifera
(accession code XP_001121759), Bombyx mori
(accession code AAY86706), and Aedes aegypti
(accession code EAT45400).
A homolog of FKBP46 was found in the EcR complex (which includes EcR, USP, and FKBP46) in the nucleus of prothoracic glands of Manduca sexta
. The developmental expression patterns of EcR and USP changed in concert with the hemolymph ecdysteroid titer, whereas the expression of FKBP46 remained constant throughout development (53
). In this study, a FKBP was identified in the nuclear proteins isolated from Drosophila
L57 cells. We did not find DmEcR and DmUSP in the purified complex, because L57 is a mutant cell line developed from D. melanogaster
Kc cells by parahomologous gene targeting to inactivate EcRB1 and EcRB2 isoforms (54
). These cells lost 90% of the 20E response (55
). The lack of USP in the complex was not known, but our two-hybrid assay in yeast and insect cells showed that both DmEcR and DmUSP interacted with FKBP, suggesting that FKBP39 may form a complex with Chd64, DmEcR, and DmUSP.
The homolog of Chd64 was also found in A. mellifera (accession code XP_392114). It is interesting that both FKBP39 and Chd64 interact with DmMet, and DmMet interacts with both DmEcR and DmUSP. Since these two DmJHRE1-binding proteins interact with proteins involved in both JH action (Met) and 20E (EcR and USP) action, it is tempting to speculate that these proteins may play an important role in cross-talk between JH and 20E.
We did not detect any specific high affinity JH binding to these two DmJHRE1-binding proteins (FKBP39 and Chd64) expressed in bacteria as GST fusion proteins or in yeast as LexA or B42 fusion proteins. Electrophoretic mobility shift assay studies showed that both of these proteins expressed in yeast can bind to DmJHRE1. Therefore, these two proteins are probably involved in cross-talk between JH and 20E by binding to DNA in complex with other transcription factors involved in JH and 20E action. Studies on developmental expression of FKBP39 and Chd64 showed peaks of mRNAs of these proteins at the end of embryonic and first and second instar larval stages. The FKBP39 and Chd64 mRNA also increased significantly in the tissues cultured in Grace’s medium supplemented with JH III for 12 h. The developmental expression and JH induction of mRNAs support the above hypothesis that these proteins may be part of multi-protein complexes that mediate cross-talk between JH and 20E during molting. However, further studies are needed to determine the role of JH in formation of these multiprotein complexes as well as to regulate the affinity of these complexes to JHRE.
The prevailing hypothesis is that JH is carried by JH-binding protein (JHBP) from the synthesis site (corpora allata), transporting through the hemolymph to the target cells. The events that occur after JH reaches target cells remain unknown. In this study, additional insights are found in the JH signal transduction pathway. In the presence of ecdysteroids (mainly 20E), the EcR heterodimerizes with other members of the nuclear receptor superfamily, noticeably with the USP and other cofactors (43
). The DmEcR/DmUSP can heterodimerize with FKBP (52
) and other cofactors and bind to the ecdysone response elements (EcRE) present in the promoter regions of ecdysteroid response genes and regulate their transcription during metamorphosis (). However, during molting, higher levels of JH plus 20E may lead to an increase in the expression of JH response genes that contain JHRE, because JH may influence the strength of the interactions of FKBP39 and Chd64 with EcR·USP·Met, and these complexes may bind to response elements such as DmJHRE1 to regulate the expression of genes containing these response elements (). Our previous studies showed that JH mediated activation of C. fumiferana
JH esterase by dephosphorylation of some unidentified nuclear proteins, and protein kinase C-mediated phosphorylation prevented binding of nuclear proteins to JH-responsive promoters, resulting in suppression of JH action (16
). FKBP39 may be one of the proteins involved in binding to JHRE, depending on its phosphorylation state. Further experiments are needed to investigate the strength of these interactions between various components. Identification of proteins that bind to JH and participate in multiprotein complexes that bind to JHRE will help in understanding JH action. Studies in progress in our laboratory and other laboratories around the world should help in elucidating the action of this important hormone.
Model of the signal transduction pathway for the JH-regulated genes that contain DmJHRE1 JH response element