BIOLOGICAL OVERVIEW

The eyes absent gene, recently and more correctly termed clift, is required at an early stage in development of the compound eye. eya is not expressed in the embryonic eye primordia or in eye discs during the first larval instar, suggesting that unlike eyeless, eya is not involved in determining early commitment to eye cell fate. In fact the newly discovered mammalian eya homolog has been shown to be regulated by Pax6, the vertebrate homolog of Drosophila Eyeless (Xu, 1997). In Drosophila eya mutants, progenitor cells in the eye disc undergo programmed cell death anterior to the morphogenetic furrow, rather than proceeding along the developmental pathway leading to retinal differentiation. Thus the absence of eyes is not due to a lack of precursors but to death of those precursors. A low level of cell death normally occurs at this stage, suggesting that eya activity influences the balance between cellular differentiation and death, that is, Eya has a cell survival function. Molecular analysis identifies Eya as nuclear protein expressed in progenitor cells prior to differentiation. It thus appears that eyes absent activity is required for the survival of eye progenitor cells at a critical stage in morphogenesis (Bonini, 1993).

Besides functioning as a transcriptional co-activator that functions in conjunction with Dachshund and Sine oculis, Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceeds by means of a thiol-phosphate intermediate. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. The phosphatase function of Eya switches the function of Six1f-Dach, Eya interactors in mammals, from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival (Li, 2003; Rayapureddi, 2003; Tootle, 2003).

The fly eyes absent (eya) gene, essential for compound eye development in Drosophila, has been shown to be functionally replaceable in eye development by a vertebrate Eya homolog. The relationship between eya and that of the eyeless gene, a Pax-6 homolog, critical for eye formation in both flies and man, has been defined: eya is essential for eyeless directed eye formation (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). Directing eyeless expression to discs that generate legs, wings and the antennal region of the head generates ectopic eyes in these regions. Eya is ectopically expressed in regions where eyeless directs ectopic eye formation. eya is essential for these ectopic eyes as ectopic eyes fail to form in eya mutants. eya can itself direct ectopic eye formation, indicating that eya has the capacity to function as a master control gene for eye formation. Directing ectopic eya expression to imaginal discs induces ectopic eyeless gene expression in the antennal region of the eye-antennal disc but fails to induce ectopic eyeless expression in other discs, even though ectopic ommatidia are formed and ectopic expression of Glass occurs. eya and eyeless together are more effective in eye formation than either gene alone: when expressed together, ectopic eyes are larger and form with higher penetrance than is the case with either eyeless or eya alone; expressed togther, eye formation occurs on the genitalia, a condition never observed in individuals with either gene alone. These data indicate conservation of the pathway of eya function between flies and vertebrates; they suggest a model whereby eya/Eya gene function is essential for eye formation by eyeless/Pax-6, and that eya/Eya can in turn mediate, via a regulatory loop, the activity of eyeless/Pax-6 in eye formation (Bonini, 1997).

eyes absent also plays a role in the embryonic determination of somatic gonadal precursor (SGP) cell fate. The gonad forms from cells of two lineages: the germline and soma (see gonadogenesis and oogenesis for more information). The somatic gonadal cells generate the various cell types within the testis or ovary that support gametogenesis.

It may be useful here to provide a brief description of the origin of SGP cells: each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. Using markers for the precursors of the somatic cells of the gonad, five discrete steps have been identified in gonadal development:

1. First, SGP cells are specified within the mesoderm in parasegments 10 through 12.
2. After pole cells traverse and exit the midgut they recognize and associate primarily with specific mesodermal cells laterally positioned in the mesoderm of parasegments 11 and 12. These are the migratory gonadal precursors that delaminate from the mesodermal cell sheet.
3. In a third step, gonadal precursors and pole cells migrate anteriorly, where they contact cells in parasegment 10.
4. Next, gonadal precursors and pole cells arrest migration at parasegment 10.
5. Finally, the mesodermal cells partially ensheath the arriving cells, and the cluster coalesces into the gonad (Boyle, 1995).

The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior gonadal precursor fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. The proper regional identities, as established by homeotic gene function, are required for the arrest of migration at the correct position. abd A is required in a population of cells within parasegments 10 and 11 that partially ensheath the coalescing gonad. Mutations in iab-4, a distal enhancer element, abolish expression of abd A within these cells, blocking the coalescence of the gonad (Boyle, 1995).

clift has been identified as a regulator of Drosophila gonadogenesis. When cloned, clift turned out to be identical to eyes absent. Mutations in clift abolish gonad formation and produce female sterility. In addition to showing an eye and gonad phenotype, some clift alleles show reduced or absent ocelli and abnormal morphology of the adult brain (Nusslein-Volhard, 1984). Just as with abdominal A, clift is expressed within SGP as these cells first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in gonadogenesis. In abdominal A mutants, clift fails to be expressed, and in abd A overexpressors, clift is expressed ectopically. Despite the early expression of clift, SGP's are specified in the absence of clift function. However, they fail to maintain their fate. Consequently, germ cells do not coalesce into a gonad. In addition, using clift as a marker, it has been shown that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with a gene regulatory hierarchy involving abd A within the mesoderm. While loss of wg abolishes gonadal precursors, ectopic expression expands the population such that most cells within lateral mesoderm adopt gonadal precursor fates. Initial dorsoventral positioning of somatic gonadal precursors relies on a regulatory cascade that establishes dorsal fates within the mesoderm. tinman appears to mediate the role of ectodermally expressed decapentaplegic; in tinman mutants few or no SGP cells are detected. clift expression is subsequently refined through negative regulation by bagpipe, a gene that specifies nearby visceral mesoderm. Thus, these studies identify essential regulators of gonadal precursor specification and differentiation and reveal novel aspects of the general mechanism whereby somatic gonadal cell fate is allocated within the mesoderm (Boyle, 1997).

Does clift/eyes absent have the same targets in the eye that it has in somatic gonad precursors? Is there a unified role for this gene in the two organs? In the eye, eyes absent is implicated in cell survival, while in the gonads clift is implicated in gonadal cell fate. One clift mutant, which acts as a strong allele in gonadal fate determination, nevertheless produces a transcript. Use of this mutant allowed an examination of SGP cell development in the absence of clift function. Shortly after SGP cells are specified in cli mutants there is a reduction in the number of cli expressing SGP cells, such that by germ band retraction, few SGP cells remain. The few cells that remain fail to complete their migration to the position of gonad formation (Boyle, 1997). It would therefore appear that there might be a role for clift in SGP survival in the mesoderm as there is for eya in photoreceptor precursor survival in the eye. The answer to whether there is a unified role for eya/clift in eyes and in the mesoderm awaits discovery of the target(s) for this gene.

Identification of transcriptional targets of the dual-function transcription factor/phosphatase eyes absent

Drosophila eye specification and development relies on a collection of transcription factors termed the retinal determination gene network (RDGN). Two members of this network, Eyes absent (EYA) and Sine oculis (SO), form a transcriptional complex in which EYA provides the transactivation function while SO provides the DNA binding activity. EYA also functions as a protein tyrosine phosphatase, raising the question of whether transcriptional output is dependent or independent of phosphatase activity. To explore this, microarrays together with binding site analysis, quantitative real-time PCR, chromatin immunoprecipitation, genetics and in vivo expression analysis were used to identify new EYA-SO targets. In parallel, the expression profiles of tissue expressing phosphatase mutant eya were examined, and it was found that reducing phosphatase activity did not globally impair transcriptional output. Among the targets identified by this analysis was the cell cycle regulatory gene, string (stg), suggesting that EYA and SO may influence cell proliferation through transcriptional regulation of stg. Future investigation into the regulation of stg and other EYA-SO targets identified in this study will help elucidate the transcriptional circuitries whereby output from the RDGN integrates with other signaling inputs to coordinate retinal development (Jemc, 2007).

Two general conclusions have resulted from this work: (1) the similarity in expression profiles between tissue overexpressing wild type and phosphatase-dead eya transgenes and analysis of gene expression by quantitative PCR suggest that EYA's phosphatase is not generally required for EYA transcriptional activity, although it may be required for maximal transactivation of some target genes, and (2) the short sequence (T/C/G)GA(A/T/G)A(T/C) appears to be the only recognizable motif shared among all SO binding sites. As exemplified by in vivo validation of EYA-SO-mediated regulation of the cell cycle regulator stg, further analysis of the target genes identified in this study will likely shed new light into the mechanisms underlying EYA-SO function during development (Jemc, 2007).

The main goal of this study was to identify new targets of EYA transcriptional activity. Although adult head tissue was used for overexpression experiments, an 86% success rate in confirming changes in expression of potential targets in developing Drosophila eye-antennal imaginal discs overexpressing eya supports the ability of this system to identify similar data sets in different developmental stages. Out of the ten genes upregulated by eya overexpression in both adult head tissue and eye-antennal imaginal discs, five demonstrated enrichment of endogenous SO at one or more predicted binding sites. These predicted binding regions were conserved across a minimum of two and up to nine Drosophila species, emphasizing their likely biological relevance. Two binding sites that did not demonstrate SO enrichment were not conserved across other Drosophila species, while binding sites in the remaining three genes were conserved across multiple species and could be EYA-SO targets in other tissues (Jemc, 2007).

The core sequence shared by all of these targets is (T/C/G)GA(A/T/G)A(T/C), a pared down version of the previously proposed GTAAN(T/C)NGANA(T/C)(C/G) SO binding sequence. In Drosophila EYA-SO targets, the sequence flanking the core (T/C/G)GA(A/T/G)A(T/C) has been shown to be important only in the case of the target so, and is absent in one of the two SO binding sites identified in the lz locus and the binding sites in stg were confirmed by gel shifts. While specific flanking sequences may further stabilize SO-DNA interactions, characterization of such a flanking sequence consensus awaits further analysis. Confirmation of additional targets predicted by microarray and binding site analysis should provide for further characterization of the SO binding sequence. Out of the remaining 31 potential targets, all except one have binding sites conserved across multiple Drosophila species, suggesting that additional EYA-SO targets will be confirmed within this data set (Jemc, 2007).

While none of the previously identified EYA-SO targets were included in the final list, two targets, so and lz were upregulated upon eya overexpression, although less than the two-fold cutoff. The expression of the previously identified targets hh, ato and ey was either absent or changes were not statistically significant. One explanation for this observation is that other signaling pathways required for the expression of these genes may not be activated, or, conversely, inhibitory signaling pathways could be activated in adult head tissue (Jemc, 2007).

In addition to examining expression levels of previously identified EYA-SO targets, the list of upregulated EYA-SO target candidates was compared to genes that were upregulated by ey overexpression in microarray analyses. Because ey both induces eya and so expression and is itself transcriptionally regulated by EYA-SO, one would expect to see a number of genes similarly regulated by overexpression of either ey or eya; however, as detailed below, pairwise comparisons between the current data set to lists of candidate ey targets derived from two independent array studies, reveals a surprisingly limited overlap. One study identified 371 genes with at least 1.5-fold upregulation across two array experiments, only 55 of which were similarly upregulated in both arrays. Comparison of this data set to a second more recent report of 300 candidate genes upregulated in response to ey overexpression revealed only 24 common targets. Comparison to the current list of potential eya-so targets yielded 10 shared with the first data set and 3 common to the second results. Encouragingly, despite this limited overlap, stg, a gene shown in this study to be transcriptionally regulated by eya and so, was one of the two targets consistently upregulated in all three studies (Jemc, 2007).

As additional targets are confirmed, it is important to note that EYA may also associate with transcription cofactors other than SO to regulate gene expression. Although EYA can associate with DAC, and X-ray crystallographic analysis suggests DAC can bind DNA, targets of an EYA-DAC complex or a consensus DAC binding site have not been identified. In addition, EYA also contains an engrailed homology 1 (eh1) domain, suggesting it may be able to bind to the transcriptional repressor Groucho (GRO). However, as current in vivo data only supports a role for EYA as a transactivator complexed to SO, identification of additional EYA cofactors in vivo will be necessary to explore the potential of SO-independent EYA transcriptional functions further (Jemc, 2007).

Many of the genes identified as direct EYA-SO transcriptional targets are largely uncharacterized 'CGs' whose expression patterns in the eye will have to be studied in detail to gain further insight to EYA-SO-mediated regulation, but a few have predicted or well-studied functions that may provide insight into how EYA-SO functions during normal development and how misregulation can result in disease. Most notable on this list is stg. Given that overexpression of eya and so results in overproliferation, while their loss leads to tissue reduction, EYA-SO control of stg expression provides a mechanism for how EYA-SO regulation of the cell cycle may in turn affect cell proliferation. An interesting question for future investigation is how the relatively broad expression of EYO and SO throughout the developing retina activates stg expression only in a relatively narrow stripe of cells just anterior to the morphogenetic furrow. Given the apparent complexity of stg cis-regulatory elements, a likely explanation is that EYA-SO act combinatorially with transcriptional effectors of other signaling pathways to effect this developmental precision (Jemc, 2007).

Consistent with eya and so overexpression leading to increased tissue overgrowth in Drosophila, elevated levels of Eya and Six family members have been observed in a variety of cancers. Studies of the transcriptional targets of mammalian Eya and SO/Six proteins have identified the cell cycle regulatory genes, cyclin D1 and cyclin A1, the proto-oncogene c-Myc and ezrin, a regulator of the cytoskeleton and contributor to metastasis, suggesting intermediates through which Eya and Six family genes regulate proliferation and contribute to cancer. Identification of stg as a transcriptional target of EYA and SO in Drosophila provides not only the first direct cell cycle target in Drosophila, but also suggests another target through which EYA and SO might regulate proliferation in other organisms (Jemc, 2007).

Before parallels can be drawn to how EYA-SO targets important for Drosophila retinal development might be relevant to development and disease in other organisms, it will be necessary to examine the conservation of the transcriptional regulatory circuits. However, given the predicted functions of the gene products encoded by candidate EYA-SO targets, together with knowledge of Eya-Six function in mammalian systems, it is tempting to speculate. For example, CG12030, the Drosophila homolog of the human Gale, encodes a sugar epimerase required for galactose metabolism. As metabolic abnormalities have been demonstrated to play a part in cataract formation, and mutations in eya have been observed in patients with congenital cataracts, the identification of CG12030 as an EYA-SO target suggests intermediates through with eya might function to maintain homeostasis in the eye. Mal, which encodes a molybdenum cofactor sulfurase important for ommochrome biosynthesis, is expressed in Drosophila pigment cells in the eye and would seem a logical target of the RDGN. Mutations of the human homolog of mal, HMCS, can result in renal failure and myositis, both intriguing phenotypes given the importance of Eya-Six in vertebrate kidney and muscle development. CG15879 encodes the Drosophila homolog of human SERHL2, a member of a serine hydrolase-like family predicted to regulate muscle growth, a developmental context in which Eya and Six family genes function in Drosophila and vertebrates. Lastly, CG8449 has a predicted RabGAP/TBC domain. While RabGAPs function in a variety of developmental contexts, RabGAP-like proteins have been predicted to function in phototransduction and synaptic transmission in Drosophila and mutations in RabGAP genes have been isolated in cases of Warburg Micro syndrome, a severe autosomal recessive disorder characterized by abnormalities in the eye, as well as the central nervous system and genitals, all contexts in which Drosophila eya and so are expressed. Identification of additional EYA-SO targets and the examination of the conservation of EYA-SO transcriptional regulation across homologous genes in different species will be necessary to determine how EYA and contribute to development and disease (Jemc, 2007).

Given the importance of achieving appropriate levels of gene expression during the course of development, it is not surprising that multiple signaling pathways converge to regulate common target genes at the level of transcription. For example, hh and lz are coordinately regulated by receptor tyrosine kinase (RTK) downstream effectors of the ETS family in conjunction with EYA and SO. This study has identified stg as an EYA-SO target and suggests stg transcription is also regulated Notch and Wingless (Wg) signaling, and by RTK signaling. Thus, these results suggest a mechanism by which members of the RDGN are integrated with Notch and Wg signaling to coordinate cell proliferation. Identification of additional EYA-SO targets is likely to reveal new nodes for integration of the RDGN with other signaling pathways, explaining how signaling pathways cooperate to yield specific developmental outcomes (Jemc, 2007).

The retinal determination gene eyes absent is regulated by the EGF receptor pathway throughout development in Drosophila

Members of the Eyes absent (Eya) protein family play important roles in tissue specification and patterning by serving as both transcriptional activators and protein tyrosine phosphatases. These activities are often carried out in the context of complexes containing members of the Six and/or Dach families of DNA binding proteins. eyes absent, the founding member of the Eya family is expressed dynamically within several embryonic, larval, and adult tissues of Drosophila. Loss-of-function mutations are known to result in disruptions of the embryonic head and central nervous system as well as the adult brain and visual system, including the compound eyes. In an effort to understand how eya is regulated during development, a genetic screen was carried out designed to identify genes that lie upstream of eya and govern its expression. This study identified a large number of putative regulators, including members of several signaling pathways. Of particular interest is the identification of both yan/anterior open and pointed, two members of the EGF Receptor (EGFR) signaling cascade. The EGFR pathway is known to regulate the activity of Eya through phosphorylation via MAPK. These findings suggest that this pathway is also used to influence eya transcriptional levels. Together these mechanisms provide a route for greater precision in regulating a factor that is critical for the formation of a wide range of diverse tissues (Salzer, 2010).

This report describes a genetic screen that identified factors that direct the expression of the retinal determination gene eyes absent to the developing embryonic head and eye imaginal disc. Putative regulators were identified by the loss or expansion of Eya protein distribution within the embryonic head of stage 9 loss-of-function mutants. The findings indicate multiple signaling cascades including Notch, Hedgehog, TGFβ, and the EGFR regulate eya expression. These results are consistent with previous studies identifying Hedgehog, Ras, and TGFβ as regulators of eya function in eye development. No mutations were recovered in any of known Wingless pathway members. This was slightly unexpected as Wnt signaling and eya are known to reciprocally regulate each other. This result could imply, however, that eya is regulated differently in diverse tissues (Salzer, 2010).

A screen similar to the one described in this study successfully identified the TGFβ pathway as an important upstream regulator of another retinal determination gene, dachshund. Of interest is the observation that the loss of TGFβ signaling has differential effects on eya and dac expression. In TGFβ mutant embryos ectopic dac expression was observed in cells of the visual primordium. However, eya expression remains unaffected in this tissue and is instead lost in the subsets of cells that give rise to the protocerebrum. These differential effects are interesting as eya and dac interact genetically within the retinal determination network. Therefore it seems that these regulatory relationships vary among different tissues. It also appears that the number of distinct signaling pathways that regulate eya expression outnumbers that of dac. This is unsurprising as the expression pattern of eya, when compared to dac, is considerably more dynamic, at least within the embryonic head (Salzer, 2010).

It was of particular interested to finding that mutations in spitz, argos, anterior open/yan and pointed, all members of the EGFR signaling pathway, altered the transcriptional pattern of eya. Previous work has demonstrated that the EGFR pathway post-translationally regulates Eya activity in the developing eye through phosphorylation via Ras/MAPK at two sites within the transactivation domain. Experiments in both flies and in insect cell culture indicate that phosphorylation augments, but is not absolutely essential, for either the transcriptional activation potential of Eya or for the induction of ectopic eyes in forced expression assays (Salzer, 2010).

These findings suggest that the EGFR pathway is also required to regulate eya transcription. This is consistent with findings that eya expression is lost in mago^- clones, which reduce Ras signaling (Firth, 2009). Indeed, loss of aop/yan behind the morphogenetic furrow results in the higher levels of Eya and its facultative partner So. Both proteins are required for photoreceptor cell fate specification and maintenance. Elevated levels of Eya and So proteins in yan mutant clones are consistent with roles for Yan in suppressing photoreceptor cell fate during normal development. In yan clones, Eya protein levels are activated to significantly higher levels than that of So. One possible explanation for these results is that EGFR signaling may in fact regulate eya expression but not that of so. As EGFR signaling also regulates Eya activity, in a yan clone there may be a feedback loop that ultimately results in lowered levels of Eya phosphorylation. Reduced levels of the Eya phospho-protein, while still able to stimulate so transcription, may do so at a less efficient rate thereby leading to lower levels of ectopic So protein (Salzer, 2010).

Unexpectedly, it was found that dac, a putative downstream target of the So-Eya complex, is not up regulated in yan clones. Rather, dac expression is down-regulated when yan is removed. As So-Eya is thought to positively regulate dac expression this result is somewhat puzzling. The result does suggest that dac is regulated not only by the Eya-So complex but also by other mechanisms, possible through EGFR signaling and an intermediate repressor. The So-Eya-Dac subcircuit is under complex regulatory control. This study suggests that still greater complexity exists in the form of differential regulation by signal transduction cascades both at transcriptional and post-translational levels (Salzer, 2010).

Two modes of transvection at the eyes absent gene of Drosophila demonstrate plasticity in transcriptional regulatory interactions in cis and in trans

For many genes, proper gene expression requires coordinated and dynamic interactions between multiple regulatory elements, each of which can either promote or silence transcription. In Drosophila, the complexity of the regulatory landscape is further complicated by the tight physical pairing of homologous chromosomes, which can permit regulatory elements to interact in trans, a phenomenon known as transvection. To better understand how gene expression can be programmed through cis- and trans-regulatory interactions, this study analyzed transvection effects for a collection of alleles of the eyes absent (eya) gene. Trans-activation of a promoter by the eya eye-specific enhancers is shown to be broadly supported in many allelic backgrounds, and the availability of an enhancer to act in trans can be predicted based on the molecular lesion of an eya allele. Furthermore, by manipulating promoter availability in cis and in trans, the eye-specific enhancers of eya were demonstrated to show plasticity in their promoter preference between two different transcriptional start sites, which depends on promoter competition between the two potential targets. Finally, it was shown that certain alleles of eya demonstrate pairing-sensitive silencing resulting from trans-interactions between Polycomb Response Elements (PREs), and genetic and genomic data support a general role for PcG proteins in mediating transcriptional silencing at eya. Overall, these data highlight how eya gene regulation relies upon a complex but plastic interplay between multiple enhancers, promoters, and PREs (Tian, 2019).

The proper expression of developmental genes represents a complex interplay of interactions between regulatory sequences that include enhancers, promoters, and silencers such as PREs. Growing evidence supports that regulatory interactions depend upon the positions of DNA elements in three-dimensional space and can be influenced by potential competing interactions with neighboring DNA elements. Due to the complexity of interactions in wild type animals, mutations in regulatory sequences are critical in helping understand how the interaction landscape is assembled via the roles played by individual elements. Furthermore, the study of trans-interactions in Drosophila can uncover aspects of gene regulation that are masked or otherwise challenging to understand in other contexts. Here, by examining cis- and trans-interactions between various classes of eya mutants, a hierarchical set of interactions were demonstrated that shed insight on how enhancers may choose between multiple promoter targets, and suggest an important antagonistic relationship between transcriptional activation by eya enhancers and silencing by local PREs (Tian, 2019).

The motivation of this study was in part by a desire to better understand how gene expression is influenced by trans-interactions, or transvection, in Drosophila. The data support that the 'A, B, C' allele classification put forth by Morris (1999) accurately predicts patterns of complementation via enhancer action in trans. Specifically, complementation of Class A alleles by Class B alleles is consistently higher than that by Class C alleles at both yellow and eya. In some Class B alleles of yellow, the core promoter is directly deleted or otherwise mutated, which has led to a model wherein competition by the functional cis-promoters in Class C alleles is a likely explanation for weaker transvection relative to Class B. Other Class B alleles of yellow, and the Class B eya alleles eya³ and eya⁴, are instead characterized by transposon insertions into the 5'UTR downstream of the promoter, which may cause changes in topology that alter the balance of enhancer-promoter interaction in cis vs trans, although direct evidence for this model is lacking. It should also be noted that variation in strength of enhancer action in trans can be seen within Classes; for example, the data consistently show higher levels of complementation by the Class A allele eya² relative to that by eya¹. This could reflect differences in the specific enhancer elements affected by each deletion, or, alternatively, could result from the availability of the preferred eya-B promoter on the eya² chromosome vs. its absence on the eya¹ chromosome. Similar to these observations, several alleles of Malic Enzyme (Men) would be categorized as Class B since they each carry a promoter deletion, yet they show highly varying levels of trans-activity. Thus, The ABC classes of alleles reflect rough categorizations with respect to an allele's participation in enhancer action in trans (Tian, 2019).

To date, transvection effects have been observed for relatively few genes in the Drosophila genome, although transgenic studies using diverse enhancers and genomic locations suggest that enhancer action in trans is widely supported. However, it is noted that genetic interactions consistent with enhancer action in trans have also been reported for two other RDN members, so and eyeless (ey). It may be purely coincidental that a substantial proportion of RDN genes show transvection effects, or, alternatively, somatic homolog pairing and transvection may be of particular importance to gene regulation in the developing eye. Consistent with the latter hypothesis, trans-interactions at the spineless (ss) locus were found to be critical for later fate specifications of photoreceptor types. It will be interesting to explore the potential for trans-interactions at other genes required for eye development to further test this hypothesis (Tian, 2019).

The presence of multiple eya promoters likely reflects an ancient promoter duplication event, which, like gene duplications, can lead to varying degrees of functional redundancy or sub-functionalization between the alternate TSS. Promoter duplication appears to be widespread in the Drosophila genome; genome-wide mapping of TSS shows that approximately 27% of mapped genes can initiate transcription via two or more promoters, with an average number of 1.4 promoters per gene across all mapped TSS. Comparison to genomes of other Drosophila species suggests that a promoter duplication at eya was a relatively recent event, with evidence of two TSS in the D. melanogaster, D. simulans, and D. ananassae genomes, but not in those of D. pseudoobscura or D. virilis (Tian, 2019).

Prior analyses at eya suggest that the eya-A and eya-B promoters have undergone some degree of sub-functionalization in D. melanogaster, with the eya-B promoter being active primarily within the developing eye disc and the eya-A promoter being more broadly expressed across multiple tissues. The data support the prior finding that the eya-B promoter is the preferred target of the eye-specific eya enhancers, which are primarily located just upstream of the eya-B promoter, but roughly 10 kb from the eya-A promoter. These observations suggest a simple model wherein specificity can be dictated by relative position; for some enhancers, activity may be highest on nearby promoters, with less activity on more distal promoters. However, the data support that this model is largely driven by promoter competition at the eya locus such that, in the absence of the eya-B promoter, the eya-A promoter becomes a 'preferred' target and is highly active. Interestingly, ChIP-seq analysis using antibodies to the insulator protein su(Hw) in embryos suggests the presence of an insulator element in the eya-B first intron that would be predicted to disrupt communication between the eye-specific enhancers and the eya-A promoter. Based on these observations, it is unlikely that this candidate insulator is active in the developing eye disc (Tian, 2019).

The data regarding promoter competition and enhancer-promoter proximity is consistent with prior observations where a nearby promoter is preferred to one that is more distal, and may therefore be generalizable to many enhancers. As a potential caveat in interpreting these data, two other DNA fragments that map close to the eya-A promoter support some degree of transgene expression in the late developing eye disc. These candidate enhancers are not themselves sufficient to rescue eye phenotypes, and are therefore of unknown functional relevance in vivo, but the possibility cannot be excluded that their activity changes in some way upon deletion of the eya-B promoter such that they play a role in the upregulation of eya-A transcription observed in this background. Finally, it is noted that the current genome annotation supports evidence for a third eya TSS, defining an eya-C transcript that initiates further downstream from the eya-A TSS and is predicted to produce a truncated protein product. The biological relevance of this potential promoter and its relationship to the eya-A and eya-B transcripts is as yet unclear (Tian, 2019).

The observation of pairing sensitive silencing of eya suggests a direct role for PcG genes in regulating eya expression. In support of this hypothesis, genomic data shows that eya is embedded in a domain of H3K27^me3 in cultured cells, embryos, and third instar disc tissues, and distinct peaks of PcG proteins that are characteristic of PREs are found throughout the eya locus. Furthermore, reduction in dosage of key PcG proteins, E(z) and Pc, suppresses pairing sensitive silencing of eya, providing genetic evidence for a role for PcG proteins in directly regulating eya expression (Tian, 2019).

Recently, Erceg (2017) characterized hundreds of sequences with overlapping PcG binding and enhancer activity, and showed that these fragments can act as enhancers in some cell types and as silencing PREs in others. Notably, one of the candidate PREs upstream of eya overlaps a previously characterized enhancer, and the mutations that uncover pairing-sensitive silencing affect sequences in this region. The data support a model wherein the PRE activity of this region is active in cells outside of the developing eye, silencing eya, whereas the PRE silencing activity is overcome in primordial eye cells in order for eye development to proceed. According to this model, H3K27 methylation would be reduced or suppressed by the activity of the eya enhancers in primordial eye cells, although it was not possible to observe this directly using existing ChIP-seq data derived from mixed larval tissues. Given that several candidate PREs are found across the locus, it is as yet unclear how these different sequences may cooperate and/or interact to determine the transcriptional state of eya in a given tissue (Tian, 2019).

Interestingly, several other genes of the Retinal Determination Network (RDN) are also characterized by domains of H3K27^me3 and localized regions of PcG binding in multiple cell types. Furthermore, in addition to eya, RDN genes toy and dac have been identified as having overlapping PRE and enhancer sequences, and a neuronal enhancer from the ey gene was shown to have enhancer activity in some tissues and PRE activity in others, suggesting that direct regulation by PcG proteins could be a common feature of RDN genes. According to this model, PcG proteins would maintain RDN genes in an inactive state in non-eye tissues, whereas activation of RDN gene transcription in the developing eye would rely on the coordinated removal of repressive chromatin marks and simultaneous activation of transcription. Consistent with this hypothesis, ChIP-seq analysis shows that binding of the PcG proteins Pho and Ph at the TSS of the RDN genes so and toy is higher in haltere tissues (where the RDN genes are inactive) relative to binding in eye tissue, consistent with reduced binding of PcG proteins at PREs of RDN genes in cells with active expression. However, investigations of roles for PcG proteins in the developing eye are complicated by the widespread pleiotropic effects on gene expression caused by PcG mutations combined with the deeply intertwined regulatory network that determines eye cell fates. For example, clonal loss of E(z) and Pc in cells anterior to the morphogenetic furrow can lead to reduced expression of eya and dachshund (dac), but this is likely due to misexpression of teashirt, which can act as a negative regulator of eya and dac. Similarly, seminal work by Zhu (2018) demonstrated a role for PcG proteins in maintaining eye cell fates in the developing eye via the repression of genes that would signal an alternative wing tissue fate. Furthermore, biochemical studies show that Eya protein is a binding partner for Combgap (Cg), a sequence-specific DNA-binding protein that can recruit PcG complexes to PREs, although genetic analyses show that Cg may act in opposition to other PcG complexes in the developing eye. Ultimately, a multifaceted approach involving targeted mutations of individual response elements, combined with transgenic strategies, in backgrounds with altered availability of PcG gene products will likely be required to unravel precise roles for PcG in regulating genes in the developing Drosophila eye (Tian, 2019).

GENE STRUCTURE

Bases in 5' UTR - 395 for type 1 and 210 for type II

Exons - 5 for each transcript

Bases in 3' UTR - 553

PROTEIN STRUCTURE

There are two identified cDNA sequences initiating from different promoters. The first 19 amino acids of the type I cDNA and the first 25 of the type II are generated by alternative splicing; these transcripts differ in their first exons (Bonini, 1993).

Amino Acids - 760 for type 1 and 766 for type II

Structural Domains

Comparison of the vertebrate proteins with Drosophila Eya reveals several domains of homology. The largest domain is the C-terminal portion of the protein, referred to as ED1 (for Eya homology domain1), which shows 53% identity over 271 amino acids in all the vertebrate and fly clones. The vertebrate ED1 sequences are more similar to one another than to the fly's ED1 sequence. Within the N-terminal domain, the sequences show little conservation. A short region of weak homology, ED2, occurs between the vertebrate and fly sequences in the amino terminus. In this domain, a run of spaced tyrosine residues is conserved. The fly sequence has a weak PEST protein degredation sequence; in a similar location, the mouse Eya2 sequence has a region that may serve as a weak PEST site. The fly Eya protein sequence has a consensus nuclear localization sequence at the amino terminus, and the protein is nuclear by immunocytochemistry. However, none of the vertebrate sequences show a motif indicative of a nuclear localization sequence (Zimmerman, 1997). The Drosophila protein has several potential cyclic nucleotide-dependent, protein kinase C and tyrosine kinase phosphorylation sites (Bonini, 1993)

The retinal determination (RD) gene network encodes a group of transcription factors and cofactors necessary for eye development. Transcriptional and posttranslational regulation of RD family members is achieved through interactions within the network and with extracellular signaling pathways, including epidermal growth factor receptor/RAS/mitogen-activated protein kinase (MAPK), transforming growth factor beta/DPP, Wingless, Hedgehog, and Notch. A structure-function analyses reveals novel aspects of Eyes absent (Eya) function and regulation. The conserved C-terminal Eya domain negatively regulates Eya transactivation potential, and Groucho-Sine oculis (So) interactions provide another mechanism for negative regulation of Eya-So target genes. The transactivation potential of Eya maps to an internal proline-, serine-, and threonine-rich region that includes the Eya domain 2 (ED2) and two MAPK phosphorylation consensus sites and demonstrate that activation of the RAS/MAPK pathway potentiates transcriptional output of Eya and the Eya-So complex in certain contexts. Drosophila S2 cell two-hybrid assays were used to describe a novel homotypic interaction that is mediated by Eya's N terminus. These data suggest that EYA requires homo- and hetero-typic interactions and RAS/MAPK signaling responsiveness to ensure context-appropriate RD gene network activity (Silver, 2003).

date revised: 4 December 2023 

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