EvoprintHD of eve

BIOLOGICAL OVERVIEW

Even-skipped is a transcriptional repressor of a number of genes, including engrailed (acting indirectly through paired, runt and sloppy paired) (Fujioka, 1996), fushi tarazu, Ultrabithorax and wingless. It fulfills a primary role in segmentation. Its repressive effect on fushi tarazu transciption results in an alternating pattern of FTZ and EVE in the blastoderm. EVE also assures that Wingless is confined to posterior compartments during segmentation. EVE protein forms a symmetrical pattern of seven stripes, subdividing the blastoderm. eve's pair-rule patterning, combined with the activity of other pair-rule genes, assures that even as early as the blastoderm stage, each cell of the fly has a unique identity. Such precision in the determination of cell fate is both startling and mind boggling, taking place as it does within the first three hours of development. This is both exceptionally fast and early.

Each stripe of eve is defined by a combination of maternal factors, transcription activators, and repressors, already patterned in the egg either prior to fertilization or prior to cellularization. The stripe pattern of eve transcription is governed by regional specific enhancers. The stripe two enhancer is the best documented of all Drosophila region specific enhancers, and the work to characterize it is a classic in promoter analysis (Small, 1992).

Each stripe is regulated by a stripe-specific enhancer upstream of the structural gene. For example, in the case of eve stripe 2, a minimal stripe 2 enhancer has been identified between -1.5 and -1.0 kb upstream from the eve start site. It is this site specific enhancer that assures eve transcription in the second pair rule stripe. Enhancers for other stripes have been identified as well.

The repressor Giant forms the anterior border of eve stripe 2. There are three binding sites for this transcription factor in the stripe 2 element. Krüppel, a gap gene expressed in the central portion of the blastoderm, determines the posterior border of the stripe, again by repression. Two of the three Krüppel sites overlap Bicoid activation sites, thus effectively prohibiting activation by Bicoid where Krüppel is expressed. Bicoid and Hunchback, both maternal proteins with anterior/posterior concentration gradients, serve to activate transcription (Small, 1992).

One of EVE's primary functions is regulation of segment polarity through EVE's indirect regulation of engrailed. In odd parasegments, graded expression of eve establishes the en stripes by setting the boundaries of the activator paired and the repressors runt and sloppy paired (Fujioka, 1995). Expression of en in even parasegments results from activation by Fushi tarazu (with Ftz-f1 as a cofactor). Only the most anterior cells of each ftz stripe express en and this restriction is dependent upon odd-skipped and naked.

The existence of regional transcriptional enhancers is perhaps the single most important explanation for regional specific gene transcription in Drosophila development, and therefore the single most important explanation for how regional identity is established during development.

Is it possible to estimate the number of target genes of the homeoproteins Eve and Ftz? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).

In vitro transcription experiments demonstrate that (1) Eve protein can directly repress the Ubx promoter, and (2) endogenous Eve protein binds to the Ubx gene in embryos. Genetic experiments have shown that eve represses Ubx in stage 11 embryos. However, this effect may be indirect and could be mediated via eveís effect on engrailed. Consequently, the expression of Ubx was examined in wild-type and eve^1.27 embryos at stage 5 -- a time before Engrailed protein is significantly expressed. UBX mRNA is present in four stripes in the posterior half of wild-type stage 5 embryos, with the anterior-most stripe (stripe 1) being the more prominent. In eve^1.27 mutants, Ubx expression is derepressed in a region including stripe 1 and stripe 2, reaching the same level of expression as stripe 1. Ubx expression is not significantly affected in the posterior of the embryo, either because Eve binds, but does not regulate Ubx in posterior cells, or because Eveís function is redundant with that of other transcription factors in these cells. It is also possible that Eve does not bind to Ubx in the posterior of the embryo (though this explanation is considered less likely). Whatever the reason, the early regulation of Ubx supports the evidence that Eve directly represses Ubx (Liang, 1998).

hsp70 is known to be induced uniformly in all cells when Drosophila cells are raised above 25ƒC. Hence, it initially seemed unlikely that this gene would be regulated by Eve. However, the discovery that Eve crosslinks to hsp70 with only half the strength it crosslinks to Ubx suggested that hsp70 expression should be reexamined. At stages 0-3, Drosophila embryos produce no detectable hsp70 transcripts. In older wild-type embryos, 37ƒC heat shocks for more than 1 minute induce hsp70 at relatively uniform levels in most cells, although a weak segmentally repeated pattern of transcripts is observed in embryos after stage 10. In contrast, heat shocks at 37ƒC for only 15-30 seconds induce hsp70 transcripts in a pair-rule like manner at cellular blastoderm and in segmentally repeated patterns in older embryos. In eve^1.27 embryos heat shocked for the same brief period, these patterns are not observed at stage 5 and are much less pronounced in later stages. Thus, Eve does regulate hsp70, and the crosslinking data, together with the fact that this regulation is observed soon after Eve becomes maximally expressed, strongly suggest that this regulation is at least partly direct. To estimate the degree of regulation by Eve, video microscopy was used to measure the changing intensity of stain at stage 5. Quantitation of a number of typical embryos suggests that hsp70 transcript levels vary two fold between the centers of the stripe and inter-stripe regions (Liang, 1998).

Eve crosslinks at the same levels to actin 5C and hsp70: like hsp70, actin 5C was thought to be uniformly expressed throughout most of embryogenesis. However, although maternally derived transcripts are uniformly distributed at stages 0-8, by stages 11 and 12 zygotic actin 5C transcripts are present in a series of different segmentally repeated patterns whose transcript levels vary two- to four-fold between stripe and inter-stripe regions. These patterns do not resemble those of hsp70, suggesting that they are due to specific regulation of actin 5C, and are not due to a general change in transcription of ubiquitously expressed genes. In eve mutant embryos, the initial pattern of actin 5C in the epidermis is not observed, and the later pattern in the mesoderm becomes altered. Since by stage 11 Eve has initiated a complex cascade of regulatory transcription factors and is itself expressed in only a few cells, the effect of Eve on actin 5C at this stage must be indirect. However, it remains plausible that Eve directly regulates actin 5C transcription at stage 5, but that maternal mRNAs obscure this, and that in later development other later expressed selector homeoproteins, such as En and the Hox proteins, directly control actin 5C. Certainly, this analysis of actin 5C adds to the view that more genes are spatially regulated than had been previously thought. The rosy gene is bound by Eve and Ftz with only 1/2 to 1/3 the binding strength of either hsp70 or actin 5C, when bound by Eve and Ftz. At stages 5-6, rosy transcripts are expressed in a broad ventral stripe that in most wild-type embryos show a pair-rule like pattern. The variations in transcript levels are 1.2 to 1.5 fold between the stripe and inter stripe regions. In eve^1.27 mutant embryos, these pair-rule like modulations are not observed. Therefore, rosy is downstream of eve at stage 5, and the low levels of Eve binding to this gene may be responsible for this weak regulation. The Adh gene was initially chosen for the in vivo DNA binding studies because Northern blots had indicated that this gene was not expressed at stages 5-9, and thus there was no reason to suspect that this gene might be bound by Eve or Ftz. In situ hybridization confirms that Adh transcripts are not detectable at stage 5, but, interestingly, at stage 14 Adh is expressed in a segmentally repeated pattern that suggests that it is downstream of the Hox genes. In the most simple sense then, the binding of Eve to Adh at stage 5 is probably nonfunctional. However, this binding may indicate that in later development Adh may be a direct target of other selector homeoproteins. When Adh expression is activated, these and other transcription factors may have greater access to this gene (Liang, 1998).

What percentage of genes are downstream of the selector homeoproteins? The above data suggest that the selector homeoproteins may regulate many more genes than initially assumed. To more thoroughly test this idea, the expression patterns of genes selected at random were analyzed. About 200 colonies were randomly picked from three separate plasmid cDNA libraries prepared from the mRNA of either 0-4 hour, 4-8 hour, or 8-12 hour old embryos. In situ hybridizations to whole-mount embryos were then performed using probes prepared from each clone. The DNA sequence of 99 clones from the 8-12 hour library was also determined to identify the genes encoding them. Just after fertilization (stages 0-1), a majority of genes express maternally derived transcripts that are uniformly distributed throughout the embryo. At cellular blastoderm (stages 5-6), maternally derived transcripts can still be detected at reduced levels for most genes, but some genes express zygotically derived transcripts in either pair-rule or other patterns. By stages 10-14, maternal transcripts have largely decayed, and most genes are expressed in either segmentally repeating patterns or in a relatively uniform manner. A subset of genes is more prevalent in the 0-4 hour library and, to a lesser extent, in the 4-8 hour library than in the 8-12 hour library. These highly expressed genes express maternal transcripts that perdure until after stage 8 (5.5 hours) and tend to be uniformly expressed at stages 10-14. Since the 8-12 hour library was prepared from mRNA in which these transcripts no longer predominate, it should give a better estimate of typical gene expression patterns. In support of this, the proportion of genes in the 8-12 hour library that do not have maternal contributions is most similar to that predicted from genetic experiments (i.e. 30%-40%). Also, the estimate of the proportion of zygotic and maternal mRNAs at different stages of development provided by the 8-12 hour library agrees most closely with the results of total RNA labeling experiments. These labeling experiments indicate that 10%-15% of total cytoplasmic poly(A) mRNAs are zygotically derived by stage 5 and that 89% of stage 14 transcripts are zygotic (Liang, 1998).

Taking the 8-12 hour library as most representative of Drosophila genes, a majority of genes whose zygotic transcription can be detected at stage 5 are expressed in pair-rule patterns. These patterns are in a variety of registers relative to one another and to Eve and Ftz, indicating that these patterns are generated by the combinatorial activities of maternal, gap and other pair-rule genes and do not result solely from control by Eve and Ftz. To determine what percent of genes are regulated by Eve and Ftz at stage 5, 11 genes were selected with the most pronounced pair-rule patterns from both the 4-8 hour and 8-12 hour libraries. Of these, the expression of seven clearly differs in eve mutants as compared to wild-type embryos. The expression of all but one of these seven genes also changes in an equally pronounced manner in ftz mutant embryos. The pattern of another gene expressed in pair-rule stripes does not detectably change in either eve or ftz mutant embryos. Of the remaining three genes, their pair-rule patterns are weaker, and it could not be judged if they are regulated by Eve or Ftz. The expression of three genes not expressed in pair-rule patterns was examined. The expression of these genes is not altered in eve or ftz mutant embryos at stage 5. Thus, Eve and Ftz regulate largely the same array of genes at cellular blastoderm. For several reasons it is difficult to give an exact number of genes that are downstream of Eve and Ftz at stage 5: the number of genes assayed is relatively small; there may be possible biases in genes represented in the cDNA library; redundancy or perduring maternal mRNAs may obscure Eve and Ftzís control of some genes, and weakly patterned genes could not be assayed. However, it is suggested that 25%-50% of genes transcribed at stage 5 are downstream of Eve and Ftz. Assuming that there are 13,000 genes in Drosophila and that 22% of genes are transcribed at this stage, this suggests that about 715-1,430 genes are downstream of both Eve and Ftz at this stage (Liang, 1998).

At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. Equally, the Hox genes establish the differences between segments. Thus, all genes expressed differently in each segment should be downstream of all of the Hox genes. This is indeed the case for the Hox gene Ubx. The expression of all seven segmentally expressed genes tested is regulated by Ubx. These downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins. The more strongly regulated genes include many developmental control genes such as Enhancer of split [E(spl)] , tramtrack, division abnormally delayed (dally), and Dwnt4. A high proportion of the moderate and weakly regulated genes are involved in essential cellular functions such as splicing (e.g. RNA helicases), translation (e.g. met tRNA synthetase), general signal transduction (e.g. G-protein beta13F) and cytoskeletal structure (e.g. alpha tubulin 84B). This raises the question of whether or not modest changes in the expression of essential enzymes and structural proteins are important for morphogenesis. It is argued that they probably are. 11% of the genes picked from the 8-12 hour cDNA library do not appear to be downstream of the selector homeoproteins. Most of these genes are expressed relatively uniformly in all cells. But even these genes show some differences in expression pattern. For example, clone 1.45 (Emp24 - a protein transport gene) is more strongly expressed in the salivary gland. From this analysis, few if any genes are truly uniformly expressed and almost all genes show some distinguishing or specific pattern (Liang, 1998).

Although this analysis suggests that at least 87% of genes are directly or indirectly regulated by the selector homeoproteins, the extent of regulation is not absolute. Around 50% of genes are regulated by five fold or less, and 30% of genes are regulated by two fold or less. Most recessive lethal mutations show little or no obvious mutant phenotype when heterozygous, in comparison to a wild-type copy of the same gene. This could be taken as evidence that two fold changes in gene expression are not significant. However, this assumption is not valid. Two to three fold changes in the levels of multiple proteins involved in the same process generally have important effects on cell physiology. The metabolic flux through most pathways is not controlled at a single rate limiting step, as early theories assumed. Instead, the control of flux is generally shared by many enzymes in a pathway. For this reason, large increases in flux require the activities of a number of enzymes to be raised, and cannot be accomplished by increasing the level of just one protein. In general, moderate changes in the activities of multiple enzymes in a pathway will alter flux more than a large change in the activity of a single enzyme. This point is illustrated by the obese mouse. Here, 1.5- to 3-fold increases in the activities of eight glycolytic and lipogenic enzymes lead to a profound change in the physiology of the mouse. Genetic experiments also suggest that small changes in gene expression are significant. For example, hypomorphic mutations are often enhanced by lowering the dose of another gene in the same pathway by half. Similarly, although there are only 73 known haplo-insufficient loci in Drosophila, and only a few of these are haplo-lethals, heterozygotes for deficiencies of 3% or more of the genome are lethal for almost all regions of the genome. Thus, to determine the significance of a change in a geneís expression, it is essential to consider changes that may also have occurred in the levels of other proteins. One of the processes controlled by the selector homeoproteins is cell size. A two fold change in cell volume should require modest changes in the expression of most cytoskeletal proteins, membrane proteins, enzymes etc. It ought not to require a change in the levels of chromatin binding proteins. Different cell types may have different requirements for which geneís expression must be altered during changes in cell size. Selector homeoproteins also control the number of cell divisions, the orientation of cell divisions, cell shape, cell affinities, differentiation, and cell movement. Thus, it seems entirely reasonable that changing morphology may require the coordinated, differential regulation of a large percentage of genes, often to only moderate extents (Liang, 1998 and references).

GENE STRUCTURE

Bases in 5' UTR -93

Exons - two

Bases in 3' UTR - 165

PROTEIN STRUCTURE

Amino Acids - 376

Structural Domains

The EVE homeodomain shares only 50% homology with Fushi tarazu, Engrailed and Antennapedia (Frasch, 1987).

even-skipped is a homeobox gene important in controlling segment patterning in the embryonic fruit fly. Its homeobox encodes a DNA binding domain which binds with similar affinities to two DNA consensus sequences, one AT-rich (ATTAAATTC), the other GC-rich. A crystallographic analysis of the Even-skipped homeodomain complexed to an AT-rich oligonucleotide at 2.0 A resolution is described. The structure reveals a novel arrangement of two homeodomains bound to one 10 bp DNA sequence in a tandem fashion. This arrangement suggests a mechanism for the homeoproteins' regulatory specificity. In addition, the functionally important residue Gln50 is observed in multiple conformations making direct and water-mediated hydrogen bonds with the DNA bases (Hirsch, 1995).

The minimal 57-residue repression domain is protein rich and contains a high perentage of hydrophobic amino acids (Han, 1993).

date revised: 20 November 98

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