Ecdysone-induced protein 74EF
Both in vitro and in vivo transcription assays have been used to delineate the promoter for the 6-kb E74 isoform mRNA. Sequences upstream from position -83 have little effect on the amount of RNA synthesized in vitro, using extracts prepared from Drosophila Kc tissue culture cells. Deletion of a 5'-flanking TATA consensus sequence have no effect on the accuracy of transcriptional initiation and result in an increase in RNA synthesis, suggesting removal of a repressor binding site. Surprisingly, removal of the first two nucleotides of the transcribed region still allow relatively high levels of transcription from the correct start site position. Removal of five additional nucleotides inactivate the promoter. In vitro transcription of a series of 3' deletions defined the 3' in vitro promoter boundary at position +43. Additional 5'-flanking sequences, between -181 and -83, are necessary for efficient transcription in transfected Kc tissue culture cells. Two transcription factors that interact with the E74 promoter, zeste and GAGA, were studied in DNA-binding assays. zeste binds to two sites within the E74 promoter. These sites overlap with three of the six GAGA-binding sites. The zeste- and GAGA-binding sites lie within domains identified by deletion mapping as cis-acting transcriptional control elements (Thummel, 1989).
E74A isoform promoter is deficient for a TATA-box. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including E74A. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).
Selection of a translation initiation site is thought to be determined by relative proximity to the 5' end and sequence context of a potential initiator codon. These guidelines seem insufficient to explain translation of the Drosophila E74A isoform mRNA, whose 5' untranslated region is exceptionally long (1.8 kb) and contains many AUG triplets preceding the long open reading frame. In an effort to understand how the appropriate initiator codon is chosen, a study of E74A translation was undertaken in transfected Drosophila cells. Translation of the E74A protein utilizes at least three initiator codons: two minor forms of the protein are initiated at a CUG and an AUG, while the most abundant form initiates at a CUG. This main initiator CUG appears to be in a good context; however, it lies downstream of 17 AUG codons and 24 other CUG codons, several of which are also in good contexts. Unexpected results were obtained from sequence perturbations upstream and downstream of the main CUG initiator. Creating an AUG with a good context 72 bases 5' to the main CUG has only a modest inhibitory effect on initiation frequency at that CUG. Replacing sequences 44 bases 3' to the main CUG has an inhibitory effect on its use as an initiator as well as on the CUG 72 bases further upstream. These results indicate that factors other than context and relative proximity to the 5' end must be involved in initiator codon selection and may include elements such as secondary or tertiary structure of the RNA (Boyd, 1993).
E74A protein binds to three adjacent sites in the middle of the E74 gene. The consensus sequence for E74A protein binding, determined by random-sequence oligonucleotide selection, contains an invariant purine-rich core sequence, C/AGGAA. This sequence is also present in the binding sites of two mammalian proteins that, like E74A, are related to the ets oncoprotein. Antibody staining of larval salivary gland polytene chromosomes reveals that E74A protein binds to both early and late ecdysone-inducible puffs. This study supports Ashburner's proposal that the early puffs encode site-specific DNA binding proteins that directly interact with the early and late ecdysone-inducible puffs (Urness, 1990 and references).
The early genes are a key group of ecdysone targets that function at the top of the signaling hierarchy. In the presence of ecdysone, early genes exhibit a highly characteristic rapid and powerful induction that represents a primary response. Multiple isoforms encoded by early genes then coordinate the activation of a larger group of late genes. While the general mechanism of ecdysone-dependent transcription is well characterized, it is not known whether a distinct mechanism governs the hormonal response of early genes. Previous work has found that one of the Drosophila early genes, E75, harbors multiple functional ecdysone response elements (EcREs). This study extends the analysis to Broad and E74 and has found that EcRE multiplicity is a general feature of the early genes. Since most of the EcREs within early gene loci are situated distantly from promoters, the chromosome conformation capture method was used to determine whether higher order chromatin structure facilitates hormonal activation. For each early gene chromatin loops were detected that juxtapose their promoters and multiple distant EcREs prior to ecdysone activation. These findings suggest that higher order chromatin structure may serve as an important mechanism underlying the distinct response of early genes to ecdysone (Bernardo, 2014).
The E74A isoform promoter is directly activated by ecdysone and is subsequently repressed by ecdysone-induced proteins. There is a correspondence between 74EF puff size and the accumulation of nascent transcripts on the E74A unit. These transcripts elongate at 1.1 kb/min so that this 60 kb unit acts as a timer, delaying the appearance of its mRNA by 1 hour. E74A transcription is induced in a variety of ecdysone target tisues in late third instar larvae and during each of the ecdysone pulses that mark the six stages of Drosophila development (Thummel, 1990).
The Hr46/DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. Hr46 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Hr46 directly contributes to both of these regulatory responses. Hr46 protein binds to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory proteins, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Hr46 represses E74A, and to a lesser extent E74B, and it also represses BR-C, E75A, and E78B. Hr46 activates betaFTZ-F1. Three Hr46 binding sites are present downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of Hr46 regulation. Ectopic expression of Hr46 reveals that the polytene chromosome binding pattern is of functional significance. Hr46 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. Hr46 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 (Lam, 1997).
Nascent E74A transcripts have been spliced, and a model proposed for the order of that splicing. This study provides, for the first time, direct biochemical evidence for splicing of a low-abundance cellular
RNA before transcription termination and polyadenylation (LeMaire, 1990).
Pulses of ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. An orphan nuclear receptor, Hormone-receptor-like in 78 (DHR78), functions at the top of the ecdysteroid regulatory hierarchies. DHR78 is expressed throughout development, with peaks of expression in third instar larvae and prepupae that correlate with the known ecdysteroid pulses. Consistent with this observation, DHR78 transcription can be induced by ecdysone in cultured larval organs. DHR78 protein binds to a subset of Ecdysone receptor/Ultraspiracle binding sites in vitro, suggesting that it may interact directly with the Ecdysone receptor. Cotransfection assays have supported this model by demonstrating that DHR78 can inhibit the ecdysone induction of a reporter gene. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. The expression of usp is not affected in DHR78 mutants, consistent with its relatively modest transcriptional regulation by ecdysteroids. In contrast, all other ecdysteroid-regulated transcriptional responses examined are disrupted in DHR78 mutants. The coordinate induction of EcR, E74B, and the BR-C in mid-third instar larvae is significantly reduced, and E74A is not expressed. Fbp-1, which is induced directly by ecdysone in fat bodies, is not expressed either in these mutants. Mid-third instar larval development is characterized by an ecdysteroid-triggered switch in salivary gland gene expression, from the ng genes to the Sgs glue genes. This switch fails to occur in DHR78 mutants: ng-1 is not repressed and Sgs-4 is not induced. DHR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. Ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated post-translationally. It is proposed that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis (Fisk, 1998).
Interestingly, the available evidence indicates that EcR and usp do not function in mid-third instar larvae, when DHR78 plays its critical role in gene regulation. Both EcR and Usp proteins are expressed at very low or undetectable levels in mid-third instar larvae. Recent genetic studies have led to the surprising conclusion that EcR-B1 and usp do not function at this stage of development. Studies of the puffing patterns of the polytene chromosomes in EcR-B1 mutant third instar larvae have revealed that the 2B5 and glue gene puffs are present, but the 74EF and 75B early puffs fail to form. This observation suggests that the BR-C and glue genes are induced normally in EcR-B1 mutants, but that the response to the high-titer late larval ecdysteroid pulse is selectively blocked. Mutations in usp show a similar stage-specific effect on ecdysteroid-regulated gene expression, where the mid-third instar regulatory hierarchy occurs normally but the late third instar hierarchy is blocked. It remains possible that EcR-A could perform a function in mid-third instar larvae, although its level in the salivary gland is very low, and it would have to exert this function independently of usp. On the contrary, these studies suggest that DHR78 is the critical regulator that triggers the mid-third instar regulatory hierarchy, preparing the animal for puparium formation in response to the high-titer late larval pulse of 20E (Fisk, 1998 and references).
The function of DHR78 most closely resembles EcR and usp, in that ectopic expression of either half of the ecdysone receptor has no adverse effects on development, most likely because their activity is controlled by a hormone. In this regard, it is interesting to note that DHR78 is transcribed throughout development with peaks of mRNA accumulation in response to ecdysteroid pulses. This broad expression pattern contrasts with that of other orphan receptor genes, which are only expressed for very brief temporal intervals during development. These observations indicate that DHR78 activity is regulated posttranslationally. This regulation could be imposed at a variety of different levels, including covalent modification (such as phosphorylation or glycosylation) or interaction with a cofactor. The simplest possibility, however, is that DHR78 is regulated by an as yet unidentified hormone. The identification of the putative DHR78 ligand is clearly the next critical step toward an understanding of the function of this receptor (Fisk, 1998).
EcR and E74B are potential targets of Crooked legs. In order to determine if crol functions in gene activation hierarchies during metamorphosis, the temporal patterns of transcription for a number of ecdysone primary- and secondary-response genes were examined in crol mutant animals. These include the EcR ecdysone receptor gene as well as the BR-C, E74A, E74B, E75A, E75B, DHR3 and betaFTZ-F1. E75A and E75B are two isoforms of the E75 early puff gene that encodes orphan members of the nuclear receptor superfamily. DHR3 and betaFTZ-F1 encode distinct orphan receptors, with DHR3 functioning as an inducer of ßFTZ-F1 expression in mid-prepupae. E75B inhibits this DHR3 activation function through direct heterodimerization. betaFTZ-F1, in turn, appears to function as a competence factor that facilitates the reinduction of the early genes by ecdysone in late prepupae. DHR3 is specifically expressed in early prepupae and is unaffected by crol mutations. In contrast, the other genes are all expressed at later stages and, interestingly, their transcription is selectively reduced in mid- and late crol 4418 mutant prepupae. EcR and E74B are both submaximally transcribed in crol 4418 mid-prepupae. Similarly, the peak of BR-C, E74A, E75A and E75B transcription in response to the prepupal ecdysone pulse is significantly reduced, while the earlier induction of these genes in response to the late larval ecdysone pulse is unaffected. Consistent with the stage-specificity of this mutant phenotype, a significant reduction in the transcription of the stage-specific early gene E93 is also seen. The timing of these transcriptional responses confirms that crol mutations have no effect on the duration of larval and prepupal development, but rather indicates that crol is required for the proper magnitude of ecdysone-induced gene expression in prepupae. The level of betaFTZ-F1 mRNA is also reduced in crol 4418 /Df mutants. However, crol 6470 homozygotes show only an approximate two-fold reduction in betaFTZ-F1 mRNA levels, yet the reduction in early gene transcription in these mutants is indistinguishable from that seen in crol 4418 mutants. This observation suggests that crol works independently of betaFTZ-F1 to regulate the prepupal genetic response to ecdysone (D'Avino, 1998).
The ecdysone response
hierarchy mediates egg chamber maturation during
mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific
manner while EcR expression is ubiquitous
throughout oogenesis. Decreasing or increasing the ovarian
ecdysone titer using a temperature-sensitive mutation or
exogenous ecdysone results in corresponding changes in
early gene expression. The stage 10 follicle cell expression
of E75 in wild-type, K10 and EGF receptor (Egfr) mutant
egg chambers reveals regulation of E75 by both the Egfr
and ecdysone signaling pathways. Genetic analysis
indicates a germline requirement for ecdysone-responsive
gene expression. Germline clones of E75 mutations arrest
and degenerate during mid-oogenesis and EcR germline
clones exhibit a similar phenotype, demonstrating a
functional requirement for ecdysone responsiveness during
the vitellogenic phase of oogenesis. Finally, the expression
of Drosophila Adrenodoxin Reductase increases during
mid-oogenesis and clonal analysis confirms that this
steroidogenic enzyme is required in the germline for egg
chamber development. Together these data suggest that the
temporal expression profile of E75, E74 and BR-C may be
a functional reflection of ecdysone levels and that ecdysone
provides temporal signals regulating the progression of
oogenesis and proper specification of dorsal follicle cell
fates (Buszczak, 1999).
In other insects, including the mosquito Aedes aegypti, the
transition from the previtellogenic to the vitellogenic state is
governed by ecdysone-regulated hierarchies. Aedes
egg chambers develop synchronously and remain arrested in a
previtellogenic state until the female takes a blood meal. This
triggers production of the ecdysiotropic neuoropeptide
(EDNH) that stimulates ovarian synthesis of ecdysone. The
resulting increase in the ecdysone titer leads to the expression
of E75 and controls induction and progression of vitellogenesis
and further egg development (Pierceall, 1999). In a similar fashion,
ecdysone could be regulating egg chamber progression
past stage 8 in Drosophila. Thus, the stage-specific
expression of ecdysone response hierarchies and their
control over egg chamber development may represent an
evolutionarily conserved mechanism for coordinating the
developmental processes that occur during insect oogenesis.
While the synchronous development of a cohort of eggs
under endocrine control, as seen in the mosquito, would not be
unexpected, the asynchronous progression of Drosophila
oogenesis under similar hormonal control raises an interesting
and important question. How can a presumed endocrine factor,
such as ecdysone, regulate the sequential, asynchronous
induction of these genetic regulatory programs during
Drosophila oogenesis? Stage specificity of ecdysone-responsive
gene expression could, in principle, be controlled
either at the level of competence to respond to ecdysone or at
the level of the production or availability of the hormone itself.
The results presented here suggest the latter possibility. While analysis of EcR
germline clones indicates that the receptor is required during
mid-oogenesis, the ecdysone receptor is present throughout
oogenesis. The expression of the ecdysone response genes BR-C,
E74 and E75, in contrast, is stage-specific, and varies in
conjunction with experimental manipulation of hormone titer.
These findings suggest that individual egg chambers are
exposed to different amounts of hormone as they progress
through oogenesis (Buszczak, 1999).
In order to investigate the role of ecdysone-responsive
gene expression in the ovary,
expression of three classical early ecdysone-responsive
genes, E75, E74 and BR-C were examined. In situ hybridization
revealed that the E75 and E74 genes are transcribed
in remarkably similar patterns during oogenesis. Both
E75 and E74 transcripts are first detected in region 2b
of the germarium. Expression decreases
during stages 2-4 and low levels of E75 and E74 mRNA
are again detected in stage 5-7 egg chambers.
Transcription of E75 and E74 appears to be
upregulated during stage 8 in both the germline and
soma. This expression continues to increase until stage
10B when transcription of both genes peaks in the
follicle cells and the nurse cells.
Immunofluorescent staining reveals the
presence of BR-C protein in the follicle cell nuclei
beginning between stages 5 and 6 of oogenesis. In most of the egg chambers examined, BR-C
appears to be completely absent from the germline.
However, in rare cases, low levels of expression could
be detected in the nurse cell nuclei. These observations
are consistent with a recent report that describes follicle
cell expression of BR-C mRNA (Buszczak, 1999).
The expression of E75, E74 and BR-C in egg
chambers suggests that these genes are co-regulated by a
common signal. If these early response genes are
being regulated by ecdysone, one would expect a
dependence on the ecdysone receptor. To determine whether
the ecdysone receptor is present in the ovary, egg chambers
from Canton-S females were stained using anti-EcR
antibodies. Antibody staining reveals that germline and
somatic cells express EcR protein in their nuclei.
This expression is first detected in the germarium, appears
to be slightly upregulated during stage 4 and persists until the
late stages of oogenesis. Additionally, border cells strongly
express EcR during their migration through the nurse cell
cluster. Uso has also been detected in all cells
within the ovary. Thus, both
components of the functional ecdysone receptor are present in
the germline and soma during all stages of oogenesis (Buszczak, 1999).
In Drosophila, pulses of the steroid hormone ecdysone
trigger larval molting and metamorphosis and coordinate
aspects of embryonic development and adult reproduction.
At each of these developmental stages, the ecdysone signal
is thought to act through a heteromeric receptor composed
of the EcR and USP nuclear receptor proteins. Mutations
that inactivate all EcR protein isoforms (EcR-A, EcR-B1,
and EcR-B2) are embryonic lethal, hindering analysis of
EcR function during later development. Using transgenes
in which a heat shock promoter drives expression of an EcR
cDNA, temperature-dependent rescue of
EcR null mutants has been employed to determine EcR requirements at later
stages of development. EcR is
required for hatching, at each larval molt, and for the
initiation of metamorphosis. In EcR mutants arrested prior
to metamorphosis, expression of ecdysone-responsive genes
is blocked and normal ecdysone responses of both imaginal
and larval tissues are blocked at an early stage. These
results show that EcR mediates ecdysone signaling at
multiple developmental stages and implicate EcR in the
reorganization of imaginal and larval tissues at the onset of
metamorphosis (Li, 2000).
Western analysis of extracts from rescued EcR mutants using
antibodies directed against products of five ecdysone-responsive
genes shows that expression of three early response
genes are largely (BR-C) or completely (E74A, E75B)
abolished. Expression of the early-late gene DHR3 and
the mid-prepupal response gene betaFTZ-F1 are also severely
affected. These results show that expression of ecdysone-responsive
genes early in metamorphosis is dependent on EcR.
The results confirm and extend to the whole animal earlier
findings from analysis of larval salivary gland polytene
chromosomes that transcriptional puffing of ecdysone-responsive
genes is blocked in EcR-B1 mutants. The retention of low levels of BR-C 91 and 81 kDa
products in EcR mutants is consistent with the incomplete
block to BR-C puffing previously seen in EcR-B1 mutants
(Li, 2000).
The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. When expressed under the control of a heat-inducible promoter, dsRNA interfers efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generates defects in larval molting and metamorphosis, resulting in animals that fail to pupariate or prepupae that die with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor has no effect on puparium formation, but leads to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that express either EcR or betaFTZ-F1 dsRNA show defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis.
These results confirm and extend understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila (Lam, 2000).
To test the generality of this method, attempts were made to interfere with betaFTZ-F1 function during the onset of metamorphosis. There are two reasons why betaFTZ-F1 provides a valuable additional test of this method. (1) Unlike EcR, betaFTZ-F1 exerts a stage-specific function at the onset of metamorphosis, with no apparent function at puparium formation and an essential role in providing competence for the ecdysone-triggered prepupal-pupal transition. (2) Only hypomorphic betaFTZ-F1 mutants have been studied during the onset of metamorphosis because null mutants die during early stages of development. Thus, more severe phenotypes associated with betaFTZ-F1 RNAi might provide new insights into the function of this receptor. Expression of betaFTZ-F1 dsRNA ~18 and 12 hours before puparium formation, which is identical to the double heat-shock regime used with hs-EcRi-11, results in normal puparium formation, although 37% of these animals failed to evert one (usually) anterior spiracle. The ability of these animals to pupariate is consistent with the absence of betaFTZ-F1 expression in third instar larvae as well as the absence of any effects of betaFTZ-F1 mutations on puparium formation. The majority of animals expressing betaFTZ-F1 dsRNA, however, failed to progress through the early stages of metamorphosis and died as prepupae. Sequential heat induction of betaFTZ-F1 dsRNA at 0 and 6 hours after puparium formation leads to a similar phenotype, with all animals arresting development at the prepupal stage. Although these animals display normal gas bubble formation, they fail to translocate the bubble to the anterior end, and die after several days with a prominent bubble in the middle of the body. In addition, eversion of the adult head is completely blocked and the larval mouthhooks that are normally expelled at head eversion remain attached at the anterior end of the animal. Although betaFTZ-F1 hypomorphic mutants also show defects in adult head eversion, this phenotype is more severe and more penetrant in animals that express betaFTZ-F1 dsRNA. Most betaFTZ-F1 hypomorphic mutants die as pupae with defects in head eversion and leg elongation, with some animals surviving to adulthood. The fully penetrant prepupal lethality associated with betaFTZ-F1 RNAi is likely to be due to a severe reduction in betaFTZ-F1 function, and indicates that betaFTZ-F1 is absolutely required for progression through the mid-prepupal stage (Lam, 2000).
The effects of betaFTZ-F1 dsRNA on ecdysone-inducible gene expression were examined. Similar to the kinetics of EcR dsRNA, betaFTZ-F1 dsRNA is expressed at high levels in response to heat treatment and then turned over very rapidly. Furthermore, the levels of endogenous betaFTZ-F1 mRNA are significantly reduced in these animals, consistent with their selective degradation by RNAi. Ecdysone-induced E74A transcription is significantly reduced in animals expressing betaFTZ-F1 dsRNA: E74B is not repressed, E75A fails to be expressed, and E93 is only weakly induced. The levels of EcR mRNA are similar to those of control animals although there is a slight decrease at 10 hours after puparium formation. It is likely that this reduction reflects a requirement for betaFTZ-F1 in directing this prepupal peak in EcR activity. The levels of usp mRNA are unaffected by the expression of betaFTZ-F1 dsRNA. Importantly, all of these effects on ecdysone-regulated gene expression are virtually identical to those seen in betaFTZ-F1 mutant prepupae, indicating that betaFTZ-F1 dsRNA acts as an effective and specific block to the activity of this competence factor (Lam, 2000).
Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).
The nuclear localization of E93
in larval salivary glands provided an opportunity to determine if E93
binds to the salivary gland polytene chromosomes and, if so, to
identify the sites bound by the protein. Salivary glands were
dissected 12-14 hr after puparium formation, fixed, squashed,
and photographed to acquire accurate cytology of the banding and
puffing patterns for mapping. The chromosomes were then stained with
affinity-purified E93 antibodies, and these patterns were compared
with the original set of photographs to allow accurate mapping of the
bound sites. E93 clearly binds to the polytene chromosomes in a
reproducible and site-specific manner and is consistently detected
at 65 chromosome sites, many of which contain ecdysone-regulated
genes or programmed cell death genes. Among these sites are the 74EF
and 75B early puffs, which contain the E74 and E75
ecdysone-inducible genes, as well as the 93F puff, which contains
E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and
contain the programmed cell death genes dredd, crq,
dcp-1, and drICE, respectively. The 2B5 early puff,
containing the BR-C ecdysone-inducible gene, and 75CD,
containing βFTZ-F1 and the programmed cell death genes
rpr, hid, and grim, were not bound by E93.
These data indicate that E93 may directly regulate the genes in bound
chromosome loci and may either encode a site-specific DNA binding
protein or a chromatin-associated protein that functions as a
transcriptional regulator (Lee, 2000).
The observations that E93 is essential for salivary gland cell death and that E93 protein binds to specific sites in the salivary gland polytene chromosomes suggest that E93 may regulate the transcription of target genes that function in steroid-triggered programmed cell death. If this hypothesis is true, then E93 mutations should
impact the transcription of genes that reside in salivary gland
chromosome loci bound by E93. Salivary glands were dissected from
staged late third instar larvae, prepupae, and pupae of control and
mutant animals. Total RNA extracted from these tissues was analyzed
by Northern blot hybridization. E93 mutations have little or
no effect on the timing and levels of BR-C, E74,
and E75A transcription in the salivary glands of late third
instar larvae and early prepupae. However, the level of expression of each
of these regulatory genes is significantly reduced or absent in
salivary glands 10-24 hr following puparium formation. Although the
smaller E74B transcript is induced, the larger E74A
RNA is not detected following the prepupal pulse of ecdysone. The
levels of EcR expression in late third instar larval and
prepupal salivary glands are not altered by E93 mutations,
although its timing is delayed by 4-6 hr at the prepupal to
pupal transition. Like EcR, βFTZ-F1 transcription is delayed but the level of this mRNA is not altered in E93 mutant
salivary glands. A similar delay is observed in the parental flies that were
used for mutagenesis, indicating that this effect is due to the
genetic background. The induction of
EcR and E74B in E93 mutant prepupae, as
well as the successful completion of adult head eversion, indicates
that the prepupal pulse of ecdysone occurs in these mutant animals,
signaling the prepupal-pupal transition (Lee, 2000).
Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, βFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, βFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. This study turned to embryonic development to address these two issues. It was shown that mid-embryonic expression of DHR3 and βFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 andβFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and βFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of βFTZ-F1. DHR3 and βFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and βFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade (Ruaud, 2010).
The regulatory interactions between DHR3, αFTZ-F1 and E74A/E75A that are described in this study in embryos are indistinguishable from those seen in prepupae. First, DHR3 expression in embryos is dependent on 20E signaling. Second, DHR3 mutants display reduced levels of αFTZ-F1, E74A and E75A expression at both stages in the life cycle, and αFTZ-F1 mutants have reduced levels of E74A mRNA and no detectable E75A expression. Taken together with studies that show that ectopic αFTZ-F1 is sufficient to drive maximal expression of E74A and E75A, these results indicate that DHR3 exerts its effect on these genes through its induction of αFTZ-F1 in embryos. Third, a loss of DHR3 function during embryogenesis does not eliminate αFTZ-F1 expression. This is probably due to other upstream factors that contribute to this response. One candidate for this function is the DHR4 nuclear receptor, which is coexpressed with DHR3 in both embryos and prepupae. DHR4 mutants have no effect on DHR3 expression, but display significantly reduced levels of αFTZ-F1 mRNA in prepupae. These mutants, however, have no effect on embryonic development, suggesting that DHR4 does not play a major role in αFTZ-F1 induction at this early stage in the life cycle (Ruaud, 2010).
The late larval pulse of 20E both directly and indirectly induces DHR3 and represses αFTZ-F1. Taken together with the inductive effect of DHR3 on αFTZ-F1 expression, this regulation ensures that the peak of αFTZ-F1 expression will be delayed until the proper time during development. The observation that the embryonic 20E pulse, at ~8 hours AEL, immediately precedes DHR3 expression suggests that similar regulatory interactions are acting in embryos. However, unlike prepupae, there is no known hormone peak in late embryos that could account for the coordinated induction of E74A and E75A mRNA at this time, as is known to occur in late prepupae. It is possible that these transcripts are fully dependent on trans-acting factors such as αFTZ-F1 for their expression in embryos. Alternatively, these 20E primary-response genes might be induced by a novel temporal signal that remains to be identified (Ruaud, 2010).
It is interesting to note that a similar temporal profile of DHR3, αFTZ-F1 and E74A/E75A expression is also seen in larvae. A burst of DHR3 expression in mid-second instar larvae immediately follows the peak in the 20E titer and precedes the transient expression of αFTZ-F1, which is followed by co-expression of E74A and E75A at the end of the instar. Curiously, E75A, but not E74A, is expressed at an earlier time as well, in apparent synchrony with the 20E pulse, recapitulating the timing seen in embryos. It is thus likely that a common set of regulatory interactions function in both embryos and larvae to dictate the precise timing of these expression patterns at each stage in the life cycle, prior to the third instar. Moreover, the observation that EcR, E75A and αFTZ-F1 mutants display defects in larval molting indicates that their expression is essential for proper progression through these stages in development (Ruaud, 2010).
DHR3 and αFTZ-F1 null mutations lead to fully penetrant embryonic lethality, with relatively minor and partially penetrant phenotypes reported in DHR3 mutant embryos and no phenotypic description of αFTZ-F1 mutant embryos. The studies described in this paper define both common and unique functions for these two nuclear receptors during embryogenesis. DHR3 and αFTZ-F1 null mutants both display a highly penetrant defect in air filling of the tracheal tree. In addition to this common function, αFTZ-F1 is required for the proper differentiation of the denticles in the ventral cuticle and DHR3 is required for VNC condensation. Both DHR3 and αFTZ-F1 mutants display apparently normal muscle movements at the end of embryogenesis, indicating that only some developmental responses are blocked at this stage. These processes of cuticle differentiation, tracheal air filling, muscular movements and VNC condensation represent the major developmental events that can be described in late embryos. Defects in three of these four pathways thus define a central role for DHR3 and αFTZ-F1 in late embryonic development. In addition, unlike prepupae, in which DHR3 and αFTZ-F1 mutants have essentially identical phenotypes, these studies establish independent functions for these two nuclear receptors
during development. Together with the previously identified early embryonic roles of the 20E receptor EcR in dorsal closure, head involution and midgut morphogenesis, these data indicate that each step of the 20E-induced transcriptional cascade controls sequential developmental programs during embryogenesis. Moreover, the observation that this transcriptional cascade is also required for larval molting suggests that it represents a stereotypic 20E response that is required for progression through each major transition in the life cycle (Ruaud, 2010).
Ectopic expression of wild-type αFTZ-F1 is sufficient to rescue the lethality of αFTZ-F1 mutants, but has no effect on the viability of DHR3 mutants, indicating that DHR3 exerts essential functions independently of its downstream partner. The causes of lethality in DHR3 and αFTZ-F1 mutant embryos, however, remain unclear. Strong loss-of-function mutations in the signal peptide peptidase (Spp) gene result in tracheal air-filling defects; however, Spp mutant embryos hatch normally and die as first or second instar larvae. Similarly, embryos with severe defects in VNC condensation can hatch into first instar larvae and survive to later stages of development. These results indicate that the lethality of DHR3 and αFTZ-F1 mutant embryos cannot be directly attributed to defects in these pathways. Rather, DHR3 and αFTZ-F1 may participate in a developmental checkpoint necessary to trigger the last steps of embryogenesis required for hatching and survival (Ruaud, 2010).
The microarray study revealed that a number of 20E-responsive genes are misregulated in DHR3 mutants, consistent with studies in prepupae that indicate a crucial role for DHR3 in 20E signaling. The microarray analysis also identified several genes that are involved in chitin metabolism and protein secretion, which could account for the defects in tracheal gas filling seen in DHR3 mutants. These included the chitinase genes Idgf5 (-8.6-fold) and kkv (+2.4-fold), the CBP Cht12 (+2.6-fold) and the COPII coat subunit sec13 (+2.5-fold). This study also identified a number of genes that play a role in axon guidance. Interestingly, most of these genes have dose-dependent effects, whereby either reduced or increased expression can disrupt nervous system development. Failure of DHR3 mutant embryos to express these genes at normal levels could thus contribute to the PNS defects (Ruaud, 2010).
Northern blot hybridization studies to examine the effects of DHR3 and αFTZ-F1 mutants on selected DHR3-regulated genes confirm and extend phenotypic studies of these mutants. Some genes, such as retn, E93 and kkv, display similar transcriptional responses in DHR3 and αFTZ-F1 mutants, whereas E74A and E75A are more significantly affected in αFTZ-F1 mutants and Idgf5 is selectively reduced in DHR3 mutants. These transcriptional effects support phenotypic studies and provide further evidence that DHR3 and αFTZ-F1 exert common and independent regulatory roles during embryogenesis. This conclusion is consistent with experimental and theoretical studies of gene regulatory networks, which indicate that transcriptional cascades provide an effective means of amplifying
signals and integrating multiple cues to provide specificity in biological responses. Transcriptional cascades can also direct temporal programs of successive gene expression, as observed in the formation of flagella in Escherichia coli and the specification of anteroposterior patterning in the Drosophila embryo. In addition, the DHR3-αFTZ-F1 transcriptional cascade involves nuclear receptors that could potentially act as ligand-regulated transcription factors, introducing an additional level of control by small lipophilic compounds. These observations support the proposal that the sequential expression of DHR3 and αFTZ-F1 at multiple stages of development can specify successive biological programs that promote appropriate progression through the life cycle. By combining insect endocrinology with the predictive power of genetics, the 20E-triggered transcriptional cascades in Drosophila provide an ideal context to define how a repeated systemic signal can be refined into precise stage-specific temporal responses during development (Ruaud, 2010).
DHR3 is required for VNC condensation, a terminal step in embryonic nervous system morphogenesis that is dependent on nervous system activity, glial cell function and apoptosis. In addition, previous studies have identified roles for DHR3 in PNS development. Interestingly, these functions, which are specific for DHR3 and are not shared with its direct target, αFTZ-F1, parallel the role of the mammalian DHR3 homolog RORα in brain development. RORα was initially identified as the gene associated with the spontaneous staggerer mutation in mice, which display ataxia associated with cerebellum developmental defects and degeneration. The cerebellum in staggerer mutants is dramatically smaller than in controls, containing fewer of the two major cell types: granule cells and Purkinje cells. Further investigation showed that this phenotype arises primarily from reduced expression in Purkinje cells of Sonic hedgehog (Shh), a mitogenic signal for granule cells. These data support the hypothesis that there is an evolutionarily conserved role for the ROR/DHR3 family of nuclear receptors in nervous system development and suggest that further functional studies of DHR3 may provide new insights into its ancestral functions in this pathway (Ruaud, 2010).
Developmental abnormalities observed in Cornelia de Lange syndrome have been genetically linked to mutations in the cohesin machinery. These and other recent experimental findings have led to the suggestion that cohesin, in addition to its canonical function of mediating sister chromatid cohesion, might also be involved in regulating gene expression. This study report that cleavage of cohesin's kleisin subunit in postmitotic Drosophila salivary glands induces major changes in the transcript levels of many genes. Kinetic analyses of changes in transcript levels upon cohesin cleavage reveal that a subset of genes responds to cohesin cleavage within a few hours. In addition, cohesin binds to most of these loci, suggesting that cohesin is directly regulating their expression. Among these genes are several that are regulated by the steroid hormone ecdysone. Cytological visualization of transcription at selected ecdysone-responsive genes reveals that puffing at Eip74EF ceases within an hour or two of cohesin cleavage, long before any decline in ecdysone receptor could be detected at this locus. It is concluded that cohesin regulates expression of a distinct set of genes, including those mediating the ecdysone response (Pauli, 2010).
The regulation of gene expression essential for normal animal development is largely mediated by sequence-specific transcription factors. One of the more mysterious aspects of developmentally regulated transcription concerns how transcription factors bound to remote regulatory sequences modulate transcription of genes many kilobases away while having no effect on neighboring genes. These distant factors must either slide long distances along chromatin fibers or else interact directly with those factors bound close to the start of transcription, with intervening chromatin forming a loop. Because of their proposed roles in chromatin looping, it is suspected that factors that regulate chromatin topology might have key roles in modulating transcription. One such factor is cohesin, a multisubunit complex essential for sister chromatid cohesion and necessary for mitotic chromosome segregation. Cohesin's Smc1, Smc3, and Rad21/Scc1 subunits form a three-membered ring, within which sister chromatin fibers are entrapped in a process that requires a separate cohesin loading factor composed of the Scc2 and Scc4 proteins. By entrapping unreplicated DNAs, cohesin could, in principle, hold distant sequences of the same chromatid together (in cis) using the same topological principle by which sister DNAs are held together in trans (Pauli, 2010).
Cohesin clearly functions in processes besides sister chromatid cohesion because it is associated with chromatin in most, if not all, quiescent cells and is essential for the pruning of postmitotic neurons, at least partly by regulating levels of ecdysone receptor. Whether or not cohesin regulates transcription has hitherto been investigated mainly by analyzing the effects of its depletion using RNA interference (RNAi). Depletion of its Rad21/Scc1 subunit causes 2-fold changes in expression of the H19 and IGF2 genes in HeLa cells and little or no effect on inducibility of the gene encoding Interferon-γ in T cells, despite destroying a putative loop between its enhancer and promoter sequences. In Drosophila BG3 tissue culture cells, up to 10- to 50-fold changes in the level of transcripts from the enhancer of split and invected-engrailed loci were detected 6 days after RNAi treatment. Intriguingly, substantial changes in mRNA levels for these transcripts were only observed 3 days following RNAi treatment. Though insightful, these experiments have a number of limitations. The effects on transcription are either modest or they are only seen long after cohesin depletion and might therefore be secondary effects due to chromosome missegregation, defective DNA repair, or some other hitherto-uncharacterized state of stress induced by a loss of cohesin activity (Pauli, 2010).
Another line of evidence hinting at a role for cohesin in transcriptional control is the finding that inactivation of one allele of Nipped-B, the Drosophila ortholog of Scc2, alters long-range enhancer-promoter interactions at the homeotic loci cut and Ultrabithorax (Ubx), at least when compromised by a gypsy retrotransposon. Moreover, mutating Rad21 in zebrafish reduces expression of the hematopoietic transcription factors RUNX1 and RUNX3 during development, whereas mutations in mau-2, the Caenorhabditis elegans Scc4 ortholog, cause defects in axon guidance. Particularly striking is the finding that Cornelia de Lange syndrome (CdLS), a multisystem developmental disorder, is caused (in more than 50% of cases) by haplodeficiency of NIPBL/Delangin, the human Scc2/Nipped-B ortholog. Because tissue culture cells derived from CdLS patients have apparently normal sister chromatid cohesion, dysregulated gene expression during embryonic development has been suggested as a potential cause. There are indeed minor changes in the expression of certain genes in NIPBL± mice (up to 2.5-fold) and CdLS patient-derived cell lines (up to 4-fold), but these so far do little to explain the developmental defects associated with CdLS, which could, in principle, be due to defective DNA repair at crucial stages of development (Pauli, 2010).
Ideally, an investigation of cohesin's role in transcription should aim to observe the immediate consequences of the complex's inactivation in cells that are neither undergoing mitosis nor replicating their DNA. Sister chromatid cohesion is normally destroyed at the onset of anaphase by separase-mediated cleavage of cohesin's Rad21/Scc1 α-kleisin subunit, which destroys its topological entrapment of chromatin fibers by opening the cohesin ring (Uhlmann, 2000; Gruber, 2003). This process can be reproduced in an inducible manner using tobacco etch virus protease (TEV) in strains of Drosophila melanogaster whose α-kleisin Rad21 contains TEV cleavage sites. This study describes the effect on gene expression of TEV-induced Rad21 cleavage in a nonproliferating tissue, which constitutes conclusive evidence that cohesin has a direct role in regulating transcription (Pauli, 2010).
Development of a method to cleave Rad21 with TEV protease in a time- and tissue-specific manner has enabled assessment of the immediate and long-term consequences of cohesin inactivation on transcription in third-instar salivary glands from Drosophila. This postmitotic tissue was chosen to ensure that any effects of cohesin inactivation on the transcriptional apparatus could not be attributed to indirect or knock on effects of chromosome missegregation or defective DNA repair due to the absence of cohesin's canonical function, namely sister chromatid cohesion. Despite this precaution, cohesin cleavage causes, from 24 hr onward, major changes in cellular physiology, some of which most likely reflect a general stress-related response. It cannot at this stage be ascertained whether these highly pleiotropic events are triggered by changes in gene expression that precede them or by the loss of a novel currently unknown cohesin function. In either case, the observations demonstrate that it is very difficult to attribute functions to cohesin in regulating gene expression merely by observing the long-term consequences of its inactivation. Changes in gene expression that occur only 24 hr or more after cohesin's removal from chromosomes could be secondary or tertiary events triggered by fundamental changes in cell physiology (Pauli, 2010).
The current observations reveal an additional complication in interpreting gene expression changes. Several of the genes whose expression is affected by cohesin cleavage are genes regulated by the ecdysone receptor, whose abundance declines after 8 hr, presumably because of an almost immediate, cohesin cleavage-dependent decline in its mRNA. Thus, the precipitous decline in Sgs1 mRNAs that takes place between 8 and 16 hr could be caused by the lack of ecdysone receptor and not by the lack of cohesin per se. Such phenomena could explain many late responses to cohesin inactivation (Pauli, 2010).
Given these considerations, it is clear that in order to attribute a role for cohesin in regulating a gene on the basis of changes in its expression upon cohesin inactivation, it is necessary to demonstrate a change in transcription as soon as cohesin has been removed from chromosomes and, crucially, long before any major change in cell physiology or in the concentration of other transcription regulators. Two genes stand out in this regard, namely EcR encoding the ecdysone receptor and Eip74EF encoding an ecdysone-dependent transcription factor. Eip74EF is a particularly good candidate, because heavy transcription of this gene in third-instar larvae gives rise to a cytologically visible puff. Cohesin is associated with this puff, and its removal by Rad21 cleavage is accompanied by an immediate cessation of puffing. Crucially, contraction of band 74 caused by Rad21's removal takes place several hours before any decline in ecdysone receptor associated with it. It is therefore suggested that cohesin present at Eip74EF has a direct role in maintaining transcription of the gene. There is no reason to believe that the same is not also true for EcR, though this has not observed it at a cytological level. Because transcription of most genes is unaffected by cohesin cleavage, it is striking that transcription of EcR, as well as of a direct target gene, Eip74EF, appears to be directly regulated by cohesin. Ecdysone-responsive genes in general are enriched in cohesin domains and preferentially misregulated following cohesin cleavage; this suggests a common aspect of the transcription process at these loci that renders them particularly dependent on cohesin. It is conceivable that the interplay between the core set of gene regulatory mechanisms (transcription factors, enhancers, promoters, etc.) was insufficient to achieve the precise control that was required to orchestrate the dramatic ecdysone-induced changes that occur during the larval-to-pupal metamorphosis. It is also conceivable that cohesin, because of its ability to encircle chromatin strands, was particularly suited to fulfill this role, either by facilitating interactions between distant DNA elements in cis or by its ability to slide along DNA (Pauli, 2010).
Although Eip74EF may be the best example of a gene directly regulated by cohesin, it is by no means the only candidate. Reduced puffing at its twin, the adjacent Eip75B, also occurs before any obvious decline in ecdysone receptor at this locus. Although the drop in Eip75B mRNAs that occurs 8 hr after induction of Rad21 cleavage may be due to a decline in ecdysone receptor, the more modest decrease that occurs earlier may be due to a direct effect of cohesin's dissociation from the locus. There are other genes, for example comm2, whose mRNAs decline rapidly upon cohesin cleavage, and these may also be directly regulated by cohesin. Interestingly, transcripts from at least two genes, namely ush and Mst87F, rise rapidly after cohesin cleavage, suggesting that although cohesin promotes transcription at certain genes, it exerts repression at others (Pauli, 2010).
Cohesin's canonical function is to mediate sister chromatid cohesion. It is currently thought to perform this by entrapping sister DNAs inside a tripartite ring formed by its Smc1, Smc3, and Rad21/Scc1 subunits. This raises the important question of whether cohesin regulates gene expression using a similar topological principle. With this in mind, it has been repeatedly proposed that cohesin might regulate gene expression by facilitating the formation or maintenance of loops between remote regulatory elements and promoter regions. Such loops have not been visualized directly but have instead been inferred from coprecipitation of remote DNA sequences following formaldehyde fixation. According to this somewhat indirect assay, long-term cohesin depletion reduces interaction between an enhancer at the 3' end of the H19 gene with a remote CCCTC-binding factor (CTCF) binding site that controls imprinting of the IGF2-H19 locus. Loss of the putative loop between the CTCF binding site and the H19 enhancer is thought to enable the enhancer to activate the neighboring IGF2 gene. Cohesin depletion also disrupts a similar type of long-range interaction between distant (cohesin-associated) CTCF sites at the INFG locus, though in this case, cohesin depletion has little effect on inducibility of the locus by cytokine. The observation that cohesin in Drosophila, unlike its enrichment at CTCF binding sites in human cells, is associated with large domains raises the possibility that it can also regulate transcription by means other than the formation of loops between remote regulatory elements. By entrapping DNAs inside rings capable of sliding along chromatin, cohesin complexes may provide a potentially mobile platform for the stable association of other factors necessary for regulating (positively or negatively) the movement of polymerases through transcription units. Cohesin's intriguing potential to modulate chromatin, together with its binding to regions covering several transcription units, is seemingly at odds with the finding that differentially expressed genes are not clustered in the genome. Whatever the activity is that cohesin brings along, the data suggest that its absence affects only a subset of genes that are normally exposed to it. The identification of ecdysone-responsive genes as a class of cohesin-dependent genes highlights that there might exist still-unknown common determinants or gene-specific regulators that render a gene susceptible to changes in cohesin binding (Pauli, 2010).
In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and βFtz-f1, representing a model for steroid hormone signalling. This study has combined genetic and imaging approaches to investigate the precise role of this signalling cascade within the prothoracic gland (PG), where ecdysone synthesis takes place. These receptors operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and βFtz-f1 act early in development through a forward process to moderate DHR3 levels. These findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics (Parvy, 2014).
During the past few years, the Drosophila steroidogenic tissue has been extensively used to investigate how extrinsic and intrinsic parameters integrate to coordinate growth with developmental progression. This study shows that EcR, E75, DHR3 and βFtz-f1, which mediate ecdysone signalling, are expressed in the steroidogenic cells and are required for ecdysone synthesis during Drosophila larval development. These findings are consistent with previous studies showing that E75A mutation and βFtz-f1 disruption in the PG induce developmental arrest as a consequence of steroid deficiency. During the past few years, the Drosophila steroidogenic tissue has been extensively used to investigate how extrinsic and intrinsic parameters integrate to coordinate growth with developmental progression. This study shows that EcR, E75, DHR3 and βFtz-f1, which mediate ecdysone signalling, are expressed in the steroidogenic cells and are required for ecdysone synthesis during Drosophila larval development. These findings are consistent with previous studies showing that E75A mutation and βFtz-f1 disruption in the PG induce developmental arrest as a consequence of steroid deficiency signal, while other cells do not respond to this signal. In the case of 20E responsiveness, the gap for acquisition of competence is associated with low ecdysone titres and high βFtz-f1 levels. These findings support the notion that βFtz-f1 also acts as a competent factor for ecdysone biogenesis through a step-forward moderation of DHR3 expression. In addition, as both EcR and DHR3 knockdown also act through an early induced event and affect βFtz-f1 expression, it is conceivable that they participate in the competence acquisition through βFtz-f1. Although the molecular mechanisms that link βFtz-f1 and EcR to DHR3 must still be elucidated, this study reveals that the response following the L2 ecdysone peak is necessary to confer competence for ecdysone biogenesis at the late L3 stage by delaying the DHR3-mediated repression of steroidogenic enzymes (Parvy, 2014).
In summary, this study unravels an autoregulatory mechanism in cyclic ecdysone production. This autoregulation is likely to be coordinated with the processes that adjust ecdysone biogenesis at the L3 stage in response to environmental cues. These include nutrition, insulin signalling and the circadian rhythm that integrates through the prothoracicotropic hormone (PTTH). Interestingly, a downstream effector of PTTH is the NR DHR4, also shown to modulate the ecdysone response at the onset of metamorphosis. However, the fact that DHR4 mutants are not arrested earlier than the prepupal stage, while each RNAi tested in this study provokes a significant arrest at larval stages, suggests that DHR4 acts through an independent mechanism. Moreover, as intermediates of the nutrient, insulin and PTTH signalling interact with NRs that respond to 20E, this study provides a framework to further investigate how environmental parameters integrate with the autoregulation of cyclic ecdysone production (Parvy, 2014).
Northern blot analysis of RNA isolated from staged animals or cultured organs was used to characterize the effects of ecdysone on E74 transcription. Ecdysone directly activates both E74A and E74B promoters. E74B mRNA precedes that of E74A, each mRNA appearing with delay times that agree with their primary transcript lengths. The earlier appearance of E74B transcripts is enhanced by its activation at an approximately 25-fold lower ecdysone concentration than E74A. E74B is further distinguished from E74A by its repression at a significantly higher ecdysone concentration than that required for its induction, close to the concentration required for E74A activation. These regulatory properties lead to an ecdysone-induced switch in E74 expression, with an initial burst of E74B transcription followed by a burst of E74A transcription. The patterns of ecdysone-induced E74A and E74B transcription vary in four ecdysone target tissues. These studies provide a means to translate the profile of a hormone pulse into different amounts and times of regulatory gene expression that, in turn, could direct different developmental responses in a temporally and spatially regulated manner (Karim, 1991).
The ETS domain DNA-binding protein encoded by the E74A early gene directly induces L71-6 late gene transcription. There are four strong E74A binding sites within the 5' region of L71-6, and these sites are essential for proper L71-6 induction at puparium formation. There is a direct link between a steroid-induced transcription factor and the activation of a secondary-response promoter, indicating that steroid signals in higher organisms can be transduced and amplified through regulatory hierarchies (Urness, 1995).
Recessive loss-of-function mutations have been identified in the early gene E74, a member of the ets protooncogene family that encodes two related DNA-binding proteins, E74A and E74B. These mutations cause defects in pupariation and pupation, and result in lethality during metamorphosis. The effects of these mutations on the transcription of over 30 ecdysone-regulated genes were examined. The transcription of most ecdysone primary-response genes during late larval and prepupal development is unaffected by the E74 mutations. Rather, E74 is necessary for the appropriate regulation of many ecdysone secondary-response genes. E74B is required for the maximal induction of glue genes in mid third instar larval salivary glands, while E74A is required in early prepupae for the proper timing and maximal induction of a subset of late genes. E74A mutations affect both the timing and levels of each L71 gene. E74B mutants prematurely induce L71 in late third instar larvae; transcription continues for 2 hours longer than normal. The window of IMP-L1 transcription, which is detected in the control genotype between 2 and 8 hours after puparium formation, is expanded in both E74 mutants. E74 mutants also affect the expression of Edg78E, Edg84A, Dopa decarboxylase and Glucose dehydrogenase. E74 activity is also necessary for the correct regulation of genes expressed predominantly in the fat body, epidermis or imaginal discs. Dopa decarboxylase and Glucose dehydrogenase are expressed primarily in the epidermis, and IMP primary- and secondary-response genes are expressed primarily in the imaginal discs. These observations confirm that E74 plays a critical role in regulating transcription during the early stages of Drosophila metamorphosis. In addition, the widespread effects of the E74 mutations on transcription indicate that E74 functions in regulatory hierarchies not only in the larval salivary gland, but throughout the entire organism (Fletcher, 1995b).
The steroid hormone ecdysone signals the stage-specific programmed cell death of the larval salivary glands during Drosophila metamorphosis. This response is
preceded by an ecdysone-triggered switch in gene expression in which the diap2 death inhibitor is repressed and the reaper (rpr) and head involution defective (hid) death activators are induced. rpr is induced directly by the ecdysone-receptor complex through an essential response element in the rpr
promoter. The Broad-Complex (BR-C) is required for both rpr and hid transcription, while E74A is required for maximal levels of hid induction. diap2 induction is
dependent on FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This study identifies transcriptional regulators of programmed cell
death in Drosophila and provides a direct link between a steroid signal and a programmed cell death response (Jiang, 2000).
Both molecular and genetic studies have indicated that the BR-C and E74 function together in common developmental pathways during the onset of metamorphosis. It was therefore asked whether, like the BR-C, E74 might contribute to the ecdysone-triggered destruction of larval salivary glands. In support of this proposal, salivary gland cell death is significantly delayed in E74P[neo] animals. This mutation is a null allele that inactivates the E74A promoter. While salivary glands in control animals are completely destroyed by 16 hr after puparium formation, approximately 20% of E74P[neo]Df(3L)st-81k19 animals have salivary glands at 24 hr after puparium formation. This partially penetrant cell death defect suggests that rpr and hid expression may be reduced in E74A mutant salivary glands. To test this hypothesis, salivary glands were dissected from staged E74P[neo]/Df(3L)st-81k19 mutants, and rpr and hid expression was examined by Northern blot hybridization. Although rpr transcription is unaffected by the E74P[neo] mutation, the levels of hid transcription are significantly reduced. This observation indicates that E74A is required for the maximal induction of hid but not rpr (Jiang, 2000).
Therefore ecdysone-regulated transcription factors encoded by betaFTZ-F1, BR-C, E74, and E75 function together to direct a burst of the diap2 death inhibitor followed by induction of the rpr and hid death activators. It is proposed that cooperation between rpr and hid allows these genes to overcome the inhibitory effect of diap2, by precisely coordinating when the salivary glands are destroyed. Evidence that the ecdysone-receptor complex directly induces rpr transcription through an essential response element in the promoter, providing a direct link between the steroid signal and a programmed cell death response. The diap2 death inhibitor is expressed briefly in the salivary glands of late prepupae, foreshadowing the imminent destruction of this tissue. This transient expression is directed by at least three ecdysone-regulated transcription factors: betaFTZ-F1, E75A, and E75B. diap2 induction is dependent on the betaFTZ-F1 orphan nuclear receptor. This is consistent with the timing of betaFTZ-F1 expression, which immediately precedes that of diap2, as well as the known role of betaFTZ-F1 as an activator of gene expression in late prepupae (Jiang, 2000).
Body size determination is a process that is tightly linked with developmental maturation. Ecdysone, an insect maturation hormone, contributes to this process by antagonizing insulin signaling and thereby suppressing juvenile growth. This study reports that the microRNA miR-8 and its target, u-shaped (USH), a conserved microRNA/target axis that regulates insulin signaling, are critical for ecdysone-induced body size determination in Drosophila. The miR-8 level is reduced in response to ecdysone, while the USH level is up-regulated reciprocally, and miR-8 is transcriptionally repressed by ecdysone's early response genes. Furthermore, modulating the miR-8 level correlatively changes the fly body size; either overexpression or deletion of miR-8 abrogates ecdysone-induced growth control. Consistently, perturbation of USH impedes ecdysone's effect on body growth. Thus, miR-8 acts as a molecular rheostat that tunes organismal growth in response to a developmental maturation signal (Jin, 2012).
This study reveals the mechanism by which ecdysone suppresses insulin signaling and thereby decelerates larval growth. During larval development, ecdysone regulates the levels of miR-8 and its target, USH, a PI3 kinase inhibitor, through the EcR downstream pathway, and quantitative regulation of miR-8 by ecdysone leads to determining the final fly size. Since the ecdysone level is low during the larval stage not engaged in the molting process, a mild response of miR-8 increase by EcR inhibition in this stage would be expected. Notably, however, it was found that this increase in miR-8 level by EcRDN is constantly sustained throughout the third instar larval stage, which is the period of exponential growth. Interestingly, among early response genes of EcR signaling, E74 and BR-C are also persistently expressed during this period; these gene products repress miR-8 expression at the transcriptional level. Thus, throughout the third instar larval period, EcR downstream signaling keeps miR-8 (and, concomitantly, insulin signaling) under control. Because the duration of this regulation lasts several days, the effect of miR-8 modulation accumulates and manifests a significant impact on final body size. This cumulative effect accounts for the effect of ecdysone signaling on body size despite only modest changes in the miR-8 level. When EcR signaling was hampered throughout the larval stage, leading to a sustained increase of miR-8, the final body size became noticeably bigger (Jin, 2012).
This function of miR-8 in shaping body size provides a novel example in which an animal uses the inherent ability of miRNAs in fine-tuning target gene expression. Previously, several studies have shown that such a strategy has been used with miRNAs in diverse biological contexts. Specific examples include maintaining the optimal level of miRNA target proteins, which is critical for organismal survival, and setting the thresholds of target gene activity to prevent inappropriate development. The current data show the application of this type of strategy in a continuous process of organismal growth. By tuning the activity of insulin signaling, miRNAs could regulate organismal growth and ensure the attainment of appropriate body size. It is currently unclear whether similar regulatory mechanisms exist in other organisms. However, in humans and rodents, the miR-200 family of miRNAs are predominantly expressed in organs such as the pituitary, thyroid, testes, ovary, and breast, most of which are major target organs of steroid hormones. Moreover, the miR-200 family of miRNAs are significantly down-regulated by the estrogen hormone in breast cancer cells and uterus tissues, suggesting that the miR-200 family may also be controlled by steroids in mammals. It would be interesting to investigate whether a comparable regulatory axis of steroid hormone/miR-200/insulin signaling is conserved through metazoan evolution (Jin, 2012).
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