BIOLOGICAL OVERVIEW

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).

Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin

Steroid hormones act as important developmental switches, and their nuclear receptors regulate many genes. However, few hormone-dependent enhancers have been characterized, and important aspects of their sequence architecture, cell-type-specific activating and repressing functions, or the regulatory roles of their chromatin structure have remained unclear. This study used STARR-seq (Self-Transcribing Active Regulatory Region sequencing), a recently developed enhancer-screening assay, and ecdysone signaling in two different Drosophila cell types to derive genome-wide hormone-dependent enhancer-activity maps. The STARR-seq vector couples the candidates' activities to their sequences in cis, such that active enhancers transcribe themselves and are present among cellular RNAs. This setup allows the assessment of candidates independent of whether they are associated with endogenous enhancer-derived transcripts (eRNAs) and irrespective of their locations at or near promoters or transcription start sites (TSSs) or in transcribed regions (e.g., introns or exons), enabling genome-wide enhancer screens, Enhancer activation was shown to depend on cis-regulatory motif combinations that differ between cell types, and cell-type-specific ecdysone targeting can be predicted. Activated enhancers are often not accessible prior to induction. Enhancer repression following hormone treatment seems independent of receptor motifs and receptor binding to the enhancer, as was shown using ChIP-seq, but appears to rely on motifs for other factors, including Eip74. This strategy is applicable to study signal-dependent enhancers for different pathways and across organisms (Shlyueva, 2014).

In this study a quantitative genome-wide map was obtained of hormone-dependent enhancer activity using STARR-seq, a direct activity-based method for enhancer identification. The availability of hundreds of hormone-activated enhancers allowed the systematic dissection of their sequence features, revealing characteristic motif signatures that are predictive within strict cross-validation settings (i.e., when the sequences used for training and testing are strictly separated). These successful predictions mean that the motif signatures are shared across different enhancers and sufficiently general to predict previously unseen sequences not used for training (Shlyueva, 2014).

Interestingly, ecdysone-induced enhancers do not only contain the EcR motif but are also strongly enriched in motifs of putative partner TFs, which differ between cell types and are required for enhancer function. The insufficiency of the EcR to activate transcription and the strict dependence on additional cell-type-specific factors is an important prerequisite to achieve cell-type-specific transcriptional responses via combinatorial regulation. It has been observed for individual transcriptional enhancers that depend on different signaling pathways (Shlyueva, 2014).

Such strictly combinatorial function has been termed 'activator insufficiency' and proposes that ligand-activated TFs function combinatorially with tissue and cell-specific TFs that act as competence determinants and/or coactivators. In this study, the combination of STARR-seq and computational sequence analyses allowed identification of the motif combinations required for ecdysone-activated enhancer function in two different cell types without prior knowledge regarding the hormone receptor and/or putative partners (Shlyueva, 2014).

Repression of enhancer activity after ecdysone treatment appears to be independent of EcR motifs and receptor binding but seems to involve Eip74 motifs. Interestingly, Eip74 had previously been proposed to repress a subset of secondary ecdysone targets, because late puffs in salivary glands appeared larger in Eip74 mutant flies. Because the Eip74 motif mutant enhancer is also less strongly active in the absence of ecdysone, this could mean that Eip74 competes with an activator or that Eip74 activates the enhancer itself prior to treatment and is then depleted of cofactors, a phenomenon called transrepression that is known for hormone signaling pathways (Shlyueva, 2014).

Previous studies showed that hormone receptors bound predominantly to regions that were already accessible prior to treatment and suggested that the chromatin might predetermine hormone-responsive enhancers (Hurtado, 2011; John, 2011). Enhancers were also found that were activated by ecdysone signaling andopen prior to treatment (e.g., a strong enhancer in the Eip75 locus). Some of these open enhancers are already bound by the EcR, which might premark regions to prepare them for fast activation or repress them in the absence of ligand, which is an established function of the EcR. The latter -- 'default repression' -- is another hallmark of TFs and enhancers downstream of signaling pathways, which might ensure reliable regulatory switching. No evidence was found for default repression via the EcR and its motifs, because disruption of EcR motifs in several enhancers did not activate them (Shlyueva, 2014).

The majority of the ecdysone-activated enhancers (>60%) are, however, closed prior to treatment with no detectable DHS-seq signal. The discrepancy between these results and the ones for the ERa and GR above might stem from the fact that not all ERa and GR binding sites determined by ChIP-seq correspond to functional hormone-responsive enhancers . Interestingly, a recent study that considered ERa binding sites that produced eRNAs and were thus likely active (Hah, 2011) concluded that ERa can access and activate enhancers in closed chromatin (Hah, 2013). Together, the current findings that are based on directly assessing enhancer activities caution the interpretation of TF binding sites determined by ChIP: because TFs (and other proteins including GFP) can be frequently crosslinked to open chromatin, the majority of ChIP-seq signals might not correspond to active enhancers. Furthermore, it questions the validity of the frequently used categorization of TF binding sites into enhancers that are regulated positively or negatively based on the flanking genes' transcriptional responses. Even TFs that function exclusively as activators will have binding sites near downregulated genes, such that a repressive function might erroneously be assumed. For example, contrary to prior expectations, only the activating function of Oct4 appears to be required for pluripotency, and this study shows that ecdysone-mediated repression is indirect and independent of EcR binding (Shlyueva, 2014).

In summary, the use of the activity-based enhancer screening method STARR-seq allowed genome-wide identification of functional hormone-responsive enhancers. Combined with the computational dissection of sequence requirements, this approach revealed that the EcR functions together with cell-type- specific partner factors, which are required for enhancer activation. The study also establishes STARR-seq as the method of choice to screen for inducible enhancers downstream of signaling pathways. The combination of STARR-seq with sequence analyses promises to be a useful approach applicable to detect signaling-dependent enhancers and elucidate their sequence characteristics more generally for different signaling pathways and across organisms (Shlyueva, 2014).

GENE STRUCTURE

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).

date revised:12 June 2023 

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