Gene name - Ecdysone-induced protein 74EF
Synonyms - E74
Cytological map position - 74F1--74F4
Function - transcription factor
Keywords - molting
Symbol - Eip74EF
FlyBase ID: FBgn0000567
Genetic map position - 3-
Classification - ETS domain
Cellular location - nuclear
The functioning of E74A and E74B, the two protein isoforms encoded by Ecdysone-induced protein 74EF (Eip74EF), provides a paradigm of how alternate transcription factors with identical or similar DNA binding domains, and thus similar target specificities, can regulate different cell functions. These isoforms are coded for by the same gene but transcription of their mRNAs is activated from different promoters. Although the two proteins share a common C-terminal ETS DNA-binding domain, they have different N-terminal sequences. In addition, the regulatory networks established during molting illustrate the degree to which gene regulation becomes a system of dizzying complexity. These networks are beginning to be understood because of the ability to measure precisely the time of gene activation in Drosophila through observation of salivary gland polytene chromosome puffing.
The steroid hormone 20-hydroxyecdysone, acting through the Ecdysone receptor, directs Drosophila metamorphosis by activating a series of genetic regulatory hierarchies. The Eip74EF gene, coding for E74A and E74B, acts at the top of these hierarchies to coordinate the induction of target genes. Analysis of Eip74EF mutations provides a clue as to the function of the gene. E74B mutants are unable either to form a normal puparium or to evert their cephalic complexes, and die as prepupae or early pupae. In contrast, many E74A mutants survive the prepupal period and die as pharate adults. Based on these phenotypes, it is thought that E74B functions earlier in metamorphosis than E74A. The role of E74B can be explained by postulating that E74B is required for the proper functioning of the larval muscles during early morphogenesis. Contraction of larval muscles is required to shorten the body segments at puparium formation. These muscles begin to degenerate several hours after pupariation. The muscular movements are required for pupation, including gas bubble translocation, withdrawal of the prepupa to the posterior of the puparium and head sack eversion. All these functions are mediated by contraction of larval abdominal muscles. In contrast, E74A is required for puffing of late puffs (secondary-response genes) present at loci 21F, 22C, C2F, 63E, 71E, 72D 82F and 83E, all of which normally peak in size in the prepupal period (Fletcher, 1995c).
The timing of E74B and E74A expression reflects their different functions. E74B is transcribed in the late larval period. This late larval period represents the earliest time at which genes are expressed that relate to the molting hierarchy. The level of E74B is high 12 hours prior to puparium formation when E74A transcription levels are low. Later, just prior to puparium formation (five days after fertilization) E74A levels are high (Karim, 1991) and E74B levels are low. From four to eight hours after puparium formation the levels switch again, and E74B levels increase while E74A levels decline (mid-prepupal stage). From 14 to 16 hours after puparium formation E74A levels are once more high and E74B transcription levels again decline. How can it be explained what is happening here? The first peak of E74A transcription coincides with a high titer pulse of ecdysone at the end of the third larval instar. This pulse triggers puparium formation (pupariation) and initiates the prepupal stage. Following the first pulse of ecdysone, ecdysone levels decline (mid-prepupal stage). There is another small peak of ecdysone levels about half a day from the first pulse. This pulse marks the head eversion stage which is accompanied by a series of stereotyped behaviors including a sequence of muscle contractions. The last and highest burst of ecdysone takes place in the middle of the pupal stage when the pupal fly is transforming into an adult. It seems as though the highest E74A levels, found in the critical prepupal stage, are reached during periods in which ecdysone levels peak; conversely, E74B levels peak when ecdysone levels are low (Karim, 1991 and Thummel, 1995).
As far as gene activity is concerned, a great deal goes on between the end of the third instar larval period (marked by pupariation to form the prepupa) and head eversion (forming the pupa). For a summary of these events, see the formation of the adult. For example, FTZ-F1, expressed during the fifth day of fly development, is repressed by both itself and ecdysone, thus restricting its expression to the brief interval of low ecdysone titer in midprepupae. FTZ-F1 appears to provide the competence response to the "early genes" E54A, E75A, Broad Complex and E93. These early genes regulate the expression of late genes, expressed in late prepupal development and the pupal phase, beginning 5.5 days after fertilization. Thus FTZ-F1 acts as a bridge between expression of the earliest genes involved in metamorphosis (Ecdysone receptor and E75) and the late genes (Woodard, 1994 and Thummel, 1995).
E74 can regulate its own transcription, most likely through binding sites within the gene. For example, E74B is required for proper levels of E74A transcription during late prepupae, suggesting that E74B induces E74A at this stage in development (Fletcher, 1995c). The presence of three adjacent E74 binding sites in the middle of the E74 gene suggest that this regulation may be direct (Urness, 1990). Ectopic expression of E74B can partially repress the E78B and Hormone receptor-like/DHR3 (Hr46) orphan receptor genes, suggesting a role for E74B in the appropriate timing of gene expression. E78B and Hr46/DHR3 are expressed right after puparium formation. E74A is both necessary and sufficient for E78B induction, implicating E74A as a key regulator of E78B expression. As the authors realize, this result is paradoxical. How can E74A be responsible for E78B expression when E78B expression precedes E74A expression? Furthermore, E74A is not detectable when E78B transcription is induced during the larval period (Fletcher, 1997).
Consistent with studies of E74 loss-of-function mutations, it has been found that E74B is a potent repressor of late secondary-response gene transcription. Ectopic E74B completely prevents L71-1 and L71-6 induction in newly formed prepupae. Thus it is concluded that E74B is a potent repressor of late gene transcription. E74B appears to have a dual function in a single tissue during third instar larval development: as an activator of salivary gland glue gene transcription and as a repressor of late gene activity (Fletcher, 1995c).
Expression of E74A is sufficient to prematurely induce the L71-6 late gene. 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 (Urness, 1995). However, ectopic expression of both Broad (formerly known as Broad complex) and E74A activators in an E74B mutant background is not sufficient to prematurely induce all late genes, indicating that other factors contribute to late gene induction. One candidate for this essential inducer is encoded by the Broad Z1 isoform, corresponding to the rbp+ function of Broad. The rbp+ function is essential for late gene expression and exerts its effects through the direct interaction of the Z1 protein with late promoters. In addition, Broad Z1 expression overlaps that of E74A, suggesting Z1 might be available to coregulate late gene function. Coexpression of Z1 and E74A reveals that both together are still insufficient for L71-6 transcription. Another candidate gene for late gene regulation is crooked legs. crol mutants have no effect on E74A transcription in late third instar larvae, but show a delay in L71 induction in prepupae that is similar to that seen in E74A mutants. The Ecdysone receptor itself exerts negative control on the late puffs, preventing their premature induction by ecdysone (Fletcher, 1997).
It is concluded that E74A and E74B transcription are precisely coordinated by dynamic changes in ecdysone concentration. E74B is induced by a low ecdysone concentration and repressed by higher hormone concentrations. The ecdysone concentration required for 50% maximal E74B repression is similar to that required for 50% maximal E74A induction. Thus each rise in ecdysone titer directs an obligate switch in E74 isoforms. It is suggested that the presence of E74B protein counteracts Broad isoform Z1 and other activators that might be present, directly preventing late gene induction until the end of larval development when E74B is repressed and E74A is induced. The ETS DNA-binding domain shared by E74A and E74B allows these factors to oppositely regulate the same target genes with distinct temporal specificity, permitting the tight coupling of rises in ecdysone titer to the induction of secondary-response genes (Fletcher, 1997).
The most appealing aspect of this model is that most thinking about transcription factor activity focuses on the DNA binding domain of the protein, effectively ignoring the rest of it. Here the DNA binding domains are identical for E74A and E74B, but the N-terminal regions differ. This situation is very reminiscent of the situation found with HOX proteins. For HOX proteins the DNA binding domains of many of these proteins shares similar sequence characteristics, and their targets also share common sequences. However, outside the DNA binding domain, HOX protein sequences differ. Apparently, these different sequences will be the critical determinants of protein function. The divergent sequences outside the DNA binding domain of transcription factors are the scaffolding used for the assembly of other transcription factors and cofactors that determine the level of gene activity. Of interest is a similar switch between negative and positive ETS domain transcription factors, noted during Drosophila eye and ventral ectoderm development, concerning the opposing effects of yan and pointed. These observations demonstrate that the steroid-triggered switch in E74 transcription factor isoforms plays a central role in the proper timing of secondary-response gene expression (Fletcher, 1997).
The start site for E74B is inside the large intron between exons 5 and 6 of E74A. The two proteins share the terminal 3 exons. E74B has two transcripts of 4.9 kb and 6.0 kb with two transcriptional start sites (Burtis, 1990).
Bases in 5' UTR - 1891 for E74A
Exons - 8 for E74A and 4 for E74B
Bases in 3' UTR - 1615 for both isoforms
The two proteins share two-thirds of their sequence. Each protein has a different acidic domain near its respective amino terminus. A C-terminal basic region contains an 84 amino acid region that is 50% identical, with an 82 amino acid sequence located near the C-terminus of the protein encoded by the human c-ets-2 proto-oncogene. This region is also highly conserved in the c-ets-1 proto-oncogene and in the Drosophila ETS proteins Pointed and Yan (Urness, 1990).
The transcription factor E74 is one of the early genes induced by ecdysteroids during metamorphosis of Drosophila. This study reports the cloning and hormonal regulation of E74 from the tobacco hornworm, Manduca sexta (MsE74). MsE74 is 98% identical to that of D. melanogaster within the DNA-binding ETS domain of the protein. The 5'-isoform-specific regions of MsE74A and MsE74B share significantly lower sequence similarity (30%-40%). Developmental expression by Northern blot analysis reveals that, during the 5th larval instar, MsE74B expression correlates with pupal commitment on day 3 and is induced to maximal levels within 12h by low levels of 20-hydroxyecdysone (20E) and repressed by physiologically relevant levels of juvenile hormone I (JH I) (Stilwell, 2003).
Similarities of MsE74 to Drosophila E74 include hormonal regulation at the level of transcription -- either through de novo synthesis or stability of the transcripts. At the time of pupal commitment, MsE74B induction is similar to Drosophila at the mid-third-instar transition in that activation occurs in response to 20E. In Manduca, this induction is inhibited by JH at concentrations similar to that found in vivo during a larval molt. In striking contrast to Drosophila E74A regulation, MsE74A mRNA expression during the molts requires decreasing titers of ecdysteroid and is not induced at high ecdysteroid titers such as it is at pupariation in Drosophila (Stilwell, 2003).
In Manduca, MsE74A mRNA and protein are expressed mainly at the end of the larval and pupal molts as the ecdysteroid titer declines and in vitro it only appears after exposure to 20E followed by exposure to hormone-free medium. Similarly, in Drosophila during the molt to the third instar, DmE74A mRNA appears on the decline of the ecdysteroid titer. Thus, its requirement for 20E followed by its removal is similar to that for ßFTZ-F1 and for dopa decarboxylase (DDC) (Stilwell, 2003).
Yet, during the final larval instar, E74A mRNA appears in Drosophila at the peak of the ecdysteroid titer for pupariation and is induced in vitro by a high concentration of 20E. No E74A was detected in Manduca, either in vivo during the pupal molt at the peak of the ecdysteroid titer on day W2 or during exposure of pupally committed epidermis to 5 microg/ml 20E in vitro. The reason for this difference is unclear, yet other genes follow a similar paradigm. For instance, in Drosophila, DDC normally appears at the ends of the molts shortly before ecdysis, but also appears at high levels and is induced by 20E at the time of pupariation. In Manduca, declining 20E titers are necessary for induction of DDC. In Drosophila, both the Z2 isoform of Broad and 20E are required to induce the high levels at pupariation. BR-Z2 is present in Manduca epidermis at the time that the ecdysteroid titer peaks on day W2. Possibly either the BR-Z2 response element or the special upstream ecdysone response element that mediates this induction in Drosophila is lacking in the promoter of the Manduca E74 gene (Stilwell, 2003).
Different isoforms of an Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b have been isolated. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, it was determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia (Cho, 2004).
date revised: 15 May 99
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