The ecdysone-inducible E75 gene responsible for the 75B puff of Drosophila melanogaster encodes a family of proteins which are members of the steroid receptor superfamily. These proteins are believed to be involved in the regulation of ecdysone response. In order to investigate the evolutionary conservation of E75, the E75 gene of Manduca sexta has been identified. The putative DNA binding, hormone binding and amino and carboxy terminal flanking domains of Drosophila E75 gene are conserved in Manduca E75. However, due to a relative reduction in intron size and number and the absence of homopolymeric amino acid repeats, the E75 B transcription unit and protein are considerably smaller in M. sexta than in D. melanogaster. These findings have implications for the identification of critical structural features of E75 and also suggest that E75 has a conserved function and a shared ligand in Lepidoptera (Segraves, 1993).
Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, a genomic clone from Galleria mellonella has been isolated that reveals 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encodes a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence shows 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone does not encode the N-terminal zinc finger, but includes a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicate that the Galleria gene E75 encodes at least two isoforms, GmE75A and GmE75B, that differ in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlate closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appear in high amounts within 4 h of the ecdysteroid rise, then decline concurrently with the hormone titer decline. At the time of pupal ecdysis, there is another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA is induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment (Jindra, 1994).
Cultured IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily has shown that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, it is concluded that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes (Palli, 1995).
A cDNA of the spruce budworm, Choristoneura fumiferana, that shows high amino acid similarity with the deduced amino acid sequences of E75 cDNAs cloned from Manduca sexta, Galleria melonella, and Drosophila melanogaster, has been cloned and characterized. The longest open reading frame of this cDNA has 690 codons and its deduced amino acid sequence has all five domains typical of a steroid hormone nuclear receptor. The deduced amino acid sequence of this cDNA shows the highest identity with the deduced amino acid sequence of E75A cDNAs cloned from M. sexta, G. melonella, and D. melanogaster, and is therefore named Choristoneura hormone receptor 75A (CHR75A). The CHR75A cDNA probe detects a 2.6 kb mRNA that is abundant at the time of the ecdysteroid peaks during molting in the embryonic, larval and pupal stages. In the sixth instar larvae, CHR75 mRNA is detected in the epidermis, fat body and midgut, and maximum expression is observed during the prepupal peak of ecdysteroids in the hemolymph. CHR75 mRNA is induced in ecdysone treated CF-203 cells and in the midgut, fat body and epidermis of larvae that are fed the non-steroidal ecdysteroid agonist, RH-5992. In vitro transcription and translation of the CHR75A cDNA yields a 79 kDa protein that binds to the retinoic acid receptor related orphan receptor response element (Palli, 1997).
Degenerate primers were derived from the amino acid sequence in the DNA binding domain of the Drosophila ecdysone receptor (DmEcR). Several partial cDNAs were amplified from the shrimp epidermis by reverse transcription polymerase chain reaction (RT-PCR). One of these fragments shows the highest amino acid sequence homology to the insect ecdysone inducible gene E75. This partial cDNA was used as a probe to screen the swimming leg cDNA library of the shrimp, Metapenaeus ensis. A 3.6 kb cDNA clone was obtained. The longest open reading frame of this cDNA consists of 606 amino acids and its deduced amino acid sequence has all five domains typical of a nuclear receptor. The putative polyadenylation signal is located at about 400 bp 3' to the stop signal. The deduced amino acid sequence of this cDNA shows the highest identity to that of the E75A reported in Manduca sexta, Galleria melonella, Drosophila melanogaster, and Choristoneura fumiferana. Based on the amino acid sequence comparison, the shrimp nuclear receptor is considered the insect homolog of E75A. Northern blot analysis shows that the shrimp E75 is expressed in the epidermis, eyestalk and the nerve cord of the pre-molt shrimp. Moreover, E75 transcripts can be detected in the epidermal tissues of early pre-molt shrimp by in situ hybridization. To determine whether the shrimp could also express other E75s like the insects, 5' end RACE and RT-PCR were performed on epidermal cDNA of a single shrimp. Subcloning and DNA sequence determination of the PCR products confirmed the presence of two other forms of E75 (tentatively called E75C and E75D) in shrimp. By RT-PCR, different levels of E75 expression can be detected in the epidermis, nerve cord and the eyestalk of early pre-molt shrimp. In addition to the different levels of expression of the shrimp E75s in the epidermis, the pattern of their expression is also different during the molting cycle. This is the first report on the cloning of a shrimp nuclear receptor superfamily member (Chan, 1998).
The homolog of the ecdysteroid-induced transcription factor E75A in Drosophila melanogaster was cloned from the tobacco hornworm, Manduca sexta, and its developmental expression and hormonal regulation were analyzed. Both E75A and E75B mRNAs are found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20E in vitro causes the rapid, transient induction of E75A RNA with a peak at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) shows a 10-fold higher sensitivity to 20E. The presence of the protein synthesis inhibitor anisomycin has no effect on the induction but prevents the decline, indicating that E75A RNA is directly induced by 20E, but its down-regulation depends on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, causes an increased accumulation of E75A RNA throughout the culture period although the temporal pattern is unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis (Zhou, 1998).
The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes thatencode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, ecdysone-regulated gene expression has been investigated in vitellogenic mosquito ovaries and fat bodies. The homolog of the Drosophila E75 early ecdysone inducible gene has been identified in the yellow fever mosquito Aedes aegypti. As in Drosophila, the mosquito homolog, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses (Pierceall, 1999).
The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. This study shows that heme binds reversibly to the orphan nuclear receptor Rev-erbα, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbα-mediated gene repression. Thus, Rev-erbα serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism (Yin, 2007).
Tests were performed to see whether the heme-dependent recruitment of the NCoR-HDAC3 corepressor complex affected expression of circadian and metabolic Rev-erbα target genes. Consistent with biochemical findings, heme depletion significantly increased the expression of the core clock gene Bmal1, whereas hemin treatment significantly suppressed Bmal1 expression, indicating that intracellular heme concentrations might regulate this Rev-erbα target. Hemin treatment also repressed transcription of the PEPCK and G6Pase genes in human HepG2 liver cells. Conversely, heme depletion by knockdown of ALAS1 significantly induced G6Pase expression and in a manner that was reversed by addition of hemin, demonstrating the dependence of G6Pase transcription on heme concentrations. The repressive effect of heme was abrogated when the abundance of Rev-erbα was reduced by siRNA, indicating that the heme effect was Rev-erbα-dependen. Moreover, heme-dependent recruitment of NCoR and HDAC3, and a concomitant reduction in histone acetylation, was observed by ChIP at the endogenous G6Pase gene. Hemin treatment also repressed the expression of G6Pase and PEPCK in primary mouse hepatocytes and blunted production of glucose, demonstrating the metabolic relevance of heme binding to Rev-erbα (Yin, 2007).
The circadian expression of Rev-erbα is regulated both transcriptionally, by BMAL1-CLOCK and by Rev-erbα itself, as well as posttranslationally, by glycogen synthesis kinase 3β-mediated phosphorylation and stabilization. This study has demonstrated that alteration of heme modulates the interaction between Rev-erbα and the NCoR-HDAC3 corepressor complex. Heme concentrations oscillate in a circadian manner, and heme is also required by proteins that control various metabolic pathways and biological processes, making it a candidate for integrating circadian clock and metabolic systems. Heme negatively affects BMAL1-NPAS2-dependent transcription activation while enhancing Rev-erbα-mediated transcription repression, providing a potential means of maintaining the amplitude of circadian rhythms (Yin, 2007).
Expression of the gene encoding ALAS1, the rate-limiting enzyme in heme biosynthesis, increased in response to peroxisome proliferator activated receptor coactivator-1α, a regulator of mitochondriogenesis that increases flux through the Krebs cycle. This first and rate-limiting enzyme in heme biosynthesis requires succincyl CoA, a Krebs cycle intermediate. Gluconeogenesis competes with the Krebs cycle for metabolic intermediates whose depletion compromises heme biosynthesis as well as mitochondrial oxidative metabolism. The ability of Rev-erbα to function as a receptor for heme could provide a general mechanism for coordinating these processes (Yin, 2007).
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