Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators involved in the maintenance of developmental decisions. Although their function as transcriptional regulators of homeotic genes has been well documented, little is known about their effect on other target genes or their role in other developmental processes. The patterning of veins and interveins in the wing has been used as a model with which to understand the function of the trxG gene ash2 (absent, small or homeotic discs 2). ash2 is required to sustain the activation of the intervein-promoting genes net and blistered (bs) and to repress rhomboid (rho), a component of the EGF receptor (Egfr) pathway. Moreover, loss-of-function phenotypes of the Egfr pathway are suppressed by ash2 mutants, while gain-of-function phenotypes are enhanced. These results also show that ash2 acts as a repressor of the vein L2-organising gene knirps (kni), whose expression is upregulated throughout the whole wing imaginal disc in ash2 mutants and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes, and (2) keeping kni in an off state in tissues beyond the L2 vein. It is proposed that the Ash2 complex provides a molecular framework for a mechanism required to maintain cellular identities in the wing development (Angulo, 2004).
Loss of ash2 function causes differentiation of ectopic vein tissue, indicating that ash2 is required for intervein development, where it functions as an activator of the intervein-promoting genes net and bs, restricting rho expression to vein regions. In addition, the loss-of-function phenotypes of Egfr alleles are rescued in ash2 mutants, while the gain-of-function phenotypes are enhanced. Furthermore, rho mRNA exhibits an expanded expression pattern in ash2 mutant tissues. Thus, ash2 promotes the maintenance of intervein fate, either by activation of net and bs or by repression of the Egfr pathway. Since rho and bs/net expression is mutually exclusive, it cannot be determined whether the Ash2 complex interacts directly with one or all of them. However, since bs expression is inhibited by the loss-of-function of ash2 during larval and pupal stages, it can be proposed that ash2 acts as a long-term chromatin imprint of bs that is stable throughout development (Angulo, 2004).
The results in adult clones and from analysis of genetic interactions suggest that ash2 acts principally by maintaining B and D intervein regions, since the C intervein remains unaltered in ash2 mutants. This region is under the control of organising genes that respond to the Hh signal. One of these genes is kn, which prevents vein differentiation in the C intervein and is required for the expression of bs in this domain. bs expression is regulated by two enhancer elements: the boundary enhancer, which is dependent on hh and controls bs expression in the C intervein region through kn; and another enhancer dependent on Dpp activity, which controls bs expression in B and D intervein domains. Thus, the role of ash2 as a positive regulator of bs is mainly restricted to regions beyond the kn domain where the Dpp dependent bs enhancer is active (Angulo, 2004).
It has been found that some combinations of dpp alleles and mosaic clones of sal-C (spalt-major/spalt-related complex of zinc-finger transcription factors) result in elimination of B and D intervein regions, along with fusion of their flanking veins. Although the genetic interactions between ash2 and either bs or net could be the result of a synergistic failure to activate genes downstream of Dpp, the results indicate that this may not be the case because salm is expressed in the central domain of the wing pouch of those mutant combinations (Angulo, 2004).
It has been recently been shown that another trxG complex, the Brm complex, is involved in regulating wing vein development. Components of that complex interact genetically with net and bs at pupal stages to regulate the expression of rho, and the complex is specifically required in cells within and bordering L5 to mediate proper signalling. There are some key differences between the Brm complex and Ash2: (1) Ash2 maintains bs expression from the third instar stage; (2) the Ash2 complex is mainly required for interveins B and D; and (3) the enhancement or suppression phenotypes of the genetic interactions with Egfr and intervein-promoting alleles are much stronger for ash2 than for the Brm complex. Taken together, these results suggest that ash2 plays a crucial role in intervein identity and that each trxG complex acts in a specific spatiotemporal program to maintain organ identity (Angulo, 2004).
The positioning of vein tissues depends on the sal-C patterning dictated by the Dpp signalling pathway. Low levels of sal-C in the anterior compartment are required for the expression of kni-C, which triggers the differentiation of L2. Lack of ash2 activity results in downregulation of salm and upregulation of kni. Thus, it is possible that within the sal-C domain, the ectopic expression of kni is a result of low levels of salm. However, when high levels of salm or salr are maintained by ectopic activation, lack of ash2 nevertheless results in de-repression of kni. Moreover, kni is also cell-autonomously de-repressed by loss-of-function of ash2 in cells outside of the sal-C expression domain. Thus, the repression state in the whole wing must be maintained by factors other than sal-C. The kni/knirl L2-enhancer is subdivided into activation binding sites for Brk, En and the Sd/Vg complex, and repression binding sites for Sd/Vg, En, Salr and Brk. No changes were observed either in ß-gal expression from the EX-lacZ enhancer or in sd, vg, brk or en expression in clones lacking ash2. Therefore the de-repression of kni in ash2 mutant cells must be accounted for by a mechanism entirely different from that of the signal-dependent induction of L2, perhaps through another enhancer more global than that of L2 (Angulo, 2004).
The low levels of salm expression associated with ash2I1 clones may also be explained by de-repression of kni. In dorsal tracheal cells, kni/knrl activity represses salm transcription, and this repression is essential for branch formation. Similarly the establishment of the border between cells acquiring dorsal branch and dorsal trunk identity entails a direct interaction of Knirps with a salm cis-regulatory element. Also in the wing, kni and knrl are likely to refine the L2 position by positive auto-regulation of their own expression and by providing negative feedback to repress salm expression (Angulo, 2004).
It is possible that the de-repression of kni, intervein inhibition and appearance of extra vein tissues are linked events. The kni-C complex organises the development of the L2 vein by activating rho and inhibiting bs. Thus, kni-C participates in L2 morphogenesis by functioning downstream of salm and upstream of vein-intervein genes. The ectopic activation of kni by lack of ash2 could trigger intervein repression and vein activation. Indeed, ectopic activation of UAS-kni results in broad expression of rho and elimination of Bs expression in pupal wings, leading to the production of solid vein material. However, in adult clones not all ash2 mutant cells develop vein tissue. This raises the possibility that de-repressed kni may not be fully functional, since ectopic kni is often localised to the cytoplasm rather than the nucleus. Alternatively, ash2 could have independent functions in the wing, maintaining the repressed state of kni alongside maintenance of the intervein condition, by acting on different targets (Angulo, 2004).
The ash2112411 mutation can partially rescue the loss of L2 in kniri-1 mutants. This is in contrast to the observation that the L2 enhancer appears not to mediate the effect of ash2. The kniri-1 allele is a 252 bp deletion in the enhancer of L2. It has been shown, however, that it is possible to rescue the vein-loss phenotype of kniri-1 by expressing a UAS-rho transgene in L2. In addition, double mutant flies for kniri-1 and net partially rescue L2. It is therefore likely that the antagonistic effect of ash2 on rho could account for the partial rescue of L2 in kniri-1 ash2112411 wings, since rho mRNA is expressed in the rescued L2 (Angulo, 2004).
Some PcG genes are known to be required for the maintenance of kni expression domains in the embryo. It is also likely that some trxG genes or other complexes of trxG proteins, such as the Ash2 complex, may interact with repressor sequences necessary to keep kni expression in an off state beyond L2. Moreover, in a genome wide prediction screen it has been shown that kni contains PRE/TREs. Thus, it is proposed that ash2 acts as regulator of kni expression in the wing through an epigenetic mechanism of cellular memory similar to the trx-G regulation of homeotic genes, albeit that it remains to be seen whether kni is a direct or indirect target of ash2 (Angulo, 2004).
A well-studied mechanism through which to induce and preserve cell identities in wing imaginal discs is the response to gradients of the morphogen Dpp. This raises questions about the extent to which the response to Dpp occurs through concentration-dependent mechanisms or cellular memory. There is compelling evidence in favour of the existence of Dpp gradients that organise the pattern and growth of the wing imaginal disc. Dpp signalling causes a graded transcriptional regulation of brk by an interaction between the Dpp transducers and a brk morphogen-regulated silencer. Thus, brk appears to respond to direct morphogenetic signalling rather than remembering the inputs of previous developmental events. However, whereas activation of salm requires continuous signalling through the Dpp pathway, other targets of Dpp, such as omb, remember exposure to the signal. Stable regulation of other genes involved in wing development, such as kni repression, and net and bs activation, would also respond to the cellular memory conferred by epigenetic marks of the Ash2 complex. Thus, both mechanisms -- morphogen-dependant, which will be required for growth and patterning, and epigenetic, which will keep specific genes in an off or on state -- are likely to act simultaneously to maintain cellular identities within the wing (Angulo, 2004).
Because many developmental regulators are only expressed transiently during development, the function of epigenetic complexes is likely to be very dynamic. The developmental events required for the construction of the wing, as with many other morphogenetic events, cannot only rely on an on or off state of gene expression. Instead, morphogenesis is rather malleable and epigenetic marks could act as a means to facilitate, rather that fix, the preservation of developmental fates. It may well be that the epigenetic marks of the Ash2 complex allow changes in chromatin structure to assist the access of proteins that activate or repress gene expression. From an epigenetic point of view, the ultimate refinement of morphogenesis and maintenance of cellular memory will depend upon the interaction of these chromatin remodelling complexes with the factors that trigger or inhibit transcription (Angulo, 2004).
In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).
In the work presented in this study, the allele ash2I1, obtained after excision of the P-lacW transposon present in line l(3)12411, was used. It is lethal in early pupa, and homozygous larvae have reduced and abnormal imaginal discs and brain. The molecular alterations present in the ash2I1 allele are small changes (2-bp deletion and 5-bp insertion) in the fourth intron of the gene. Northern blot analysis of poly(A) RNA extracted from third instar larvae showed the presence of two transcripts (2 and 1.4 kb) with potential coding sequence in WT and only the small one in ash2I1 mutant flies. The longer transcript would account for the already described Ash2 protein, and 5'-rapid amplification of cDNA ends; results supported by in silico predictions from GENEID and GENSCAN show that the 1.4-kb transcript, identical in both WT and ash2I1, contains exons 5-8 present in the previously described ash2 transcript plus a novel 62-bp exon containing 28-bp encoding for amino acids. If translated, the resulting protein would be 350 aa in length and would lack the proline-, glutamic acid-, serine-, and threonine-rich region sequence and the putative double zinc-finger domain, also found in other trx-G proteins. Developmental Northern blot of WT flies showed that both transcripts are present at all stages of development except in early embryos, where only the long maternal transcript was detected. Because the insertion (TTAGG) detected in the fourth intron of the ash2I1 allele creates a putative splicing acceptor site that could generate a transcript containing a premature translation termination codon, it is tempting to speculate on a nonsense mediated decay of such RNA species. To confirm that the mutant behaves as a true trx-G mutant, genetic mosaics were generated in haltere and leg imaginal discs with the aid of the flipase-flipase recombination target (FLP-FRT) technique and the expression of Ultrabithorax (Ubx) was examined by immunohistochemistry. The down-regulation of Ubx accumulation in the homozygous ash2I1 tissue proves this mutant behaves as a trithorax mutant regarding homeotic function (Beltran, 2003).
To identify downstream genes of ash2 function, the population of mRNA species isolated from homozygous ash2I1 third instar larvae was compared with that of stage-matched WT. Four completely independent cDNA microarray experiments were carried out with poly(A) RNA isolated from separate extractions. The microarrays were constructed by using the ESTs from the Drosophila gene collection 1.0, which contains about one-third of the Drosophila genes. This collection lacks some genes known to be regulated by ash2 such as Ubx. A total of 5,139 cDNAs with a different FlyBase identifier were printed; 4,163 of them passed the quality filters in at least two of the experiments and could be used in the analysis. With a false discovery rate of ~0.025 and a fold change threshold of 1.75, 235 genes were identified of the 4,163 (5.6%) whose expression levels change significantly in the mutant, pointing to ash2 as a putative regulator of them. One hundred forty of these genes were positively regulated and 95 negatively regulated. Down-regulated genes include ash2 with a 1.76-fold change, a rather high value if the presence of the 1.4-kb transcript in the ash2I1 mutant is acknowledged. The differential expression levels of candidate genes was examined by performing semiquantitative RT-PCR analysis on selected genes (Beltran, 2003).
Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Focus was placed on the up-regulated gene FasII, and clonal analysis was performed on a Minute background, to assess whether the behavior of the FasII transcript observed with this ash2 mutant was also kept at the protein level in wing imaginal discs. Homozygous mutant cells show a clear up-regulation of FASII, mainly in the wing pouch area further away from the dorsoventral margin, where FASII was found to be very slightly expressed in WT wing discs. The up-regulation of FasII and other cell adhesion molecules like mfas, together with the up-regulation of the transcription factor vri, could explain some of the phenotypes previously found by clonal analysis, such as disruption of vein-intervein patterning, because it is known that preferential accumulation of specific adhesion molecules characterizes the final stages of vein differentiation. Furthermore, because FASII is involved in the development of the neural system, its pattern of expression in ash2I1 mutant brains was compared with that of WT; they present a distorted phenotype in the optic lobes as well as fasciculation defects in the ventral ganglion, a process in which FASII plays a central role. A gain-of-function screen (Kraut, 2001) identified ash2 and FasII as genes involved in the development of the neural system, further supporting a relationship between them (Beltran, 2003).
In a search for putative downstream genes for Set1, many genes involved in transcriptional regulation of growth and cell cycle control have been found (Nislow, 1997). According to the current results, ash2 also seems to act on genes that fall into these classes. For example, Eip75B, vri, jing, or HmgD could be classified in the first class (transcriptional regulation), whereas CyclinA (CycA), cutlet, Pu, mei-S332, or mus209 could do so in the second (cell cycle control). It was confirmed, by clonal analysis in wing imaginal discs, that the protein product of the down-regulated gene CycA is present to a lesser extent in mutant clones. The down-regulation of CycA can play a role in the proliferation defects observed in mutant cells when clones are generated in the imaginal discs. Furthermore, in genetic mosaics, homozygous mutant ash2I1 cells show effects on both cell differentiation and cell size. Normal wing cells develop a single hair, whereas ash2I1 cells can develop either a single or multiple hairs. In addition, the spacing between ash2I1 mutant cells is increased compared with that of WT ones, suggesting that ash2I1 mutant cells are larger (Beltran, 2003).
Preliminary promoter analysis of the regulated genes does not seem to reveal any defined and clear pattern. Although this needs further investigation, it may be difficult to find any consensus sequence, mainly because the regulated genes reflect all of the transcripts present at that moment and therefore a particular cellular status. Even though some authors have described cis-acting Trithorax Response Element sequences needed for some trx-G proteins to exert their function, it is not yet possible to discern between primary and secondary targets of Ash2 on the basis of this information. However, it can be stated that Ash2 function is required for a wide variety of biological processes during larval development (Beltran, 2003).
The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. A Drosophila PIP5K type I (skittles) has been cloned and mutations in this gene have been generated. This analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, no evidence was found for PIP5KI involvement in neural secretion (Hassan, 1998).
To determine if Brm physically interacts with other trithorax group proteins, the Brm complex was purified from Drosophila embryos and its subunit composition analyzed. The Brm complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the Brm complex are not encoded by trithorax group genes. The proteins that consistently copurify with Brm have been designated Brm-associated proteins (BAPs) and are referred to by their molecular mass in kDa (BAP45, BAP47, BAP55, BAP60, BAP74, BAP111 and BAP155). Two different purification schemes identify the same set of seven polypeptides associated with Brm (Papoulas, 1998).
Biochemical evidence is presented for the existence of two additional complexes containing trithorax group proteins: a 2 MDa Ash1 complex and a 500 kDa Ash2 complex. Based on their genetic properties, three of the best candidates for trx-G members that physically interact with Brm are Absent, small or homeotic discs 1 and 2 (Ash1 and Ash2), and Trithorax. In spite of being bona fide members of the trx-G, neither Ash1, Ash2 nor Trithorax are found to be a part of the Brm complex. Affinity-purified polyclonal antibodies against Ash1 detect three prominent bands in embryo extracts, the largest of which is 270 kDa. The predicted size of the Ash1 protein (244 kDa) and the variability in amount of the smaller bands detected in different experiments argues that the 270 kDa band represents full-length Ash1 and that the smaller bands are degradation products. Affinity-purified antibodies against ASH2 detect a single band of 94 kDa. Although the Brm, BAP45/Snr1, Ash1 and Ash2 proteins are readily detected by western blotting in whole embryo extracts, neither the Ash1 nor Ash2 proteins are detected in purified Brm complex. Similar experiments using antibodies against Trx did not yield reproducible results, presumably due to the low abundance and instability of this >350 kDa protein. An examination to see if Ash1 or Ash2 are physically associated with Brm in embryo extracts used a coimmunoprecipitation assay. Neither Ash1 nor Ash2 were found to coimmunoprecipitate with Brn. It is therefore concluded that the Ash1 and Ash2 proteins do not stably interact with the Brm complex. To determine whether Ash1 and Ash2 are components of protein complexes distinct from the Brm complex in the Drosophila embryo, the native molecular mass of both proteins was examined by gel filtration chromatography. The ASH1 protein has a native molecular mass of approximately 2 MDa. By contrast, Ash2 has an apparent native molecular mass of approximately 500 kDa. No monomeric Ash1 or Ash2 is detected in embryo extracts. It is concluded that the Drosophila embryo contains at least three distinct protein complexes containing trx-G proteins: the 2 MDa BRM complex, a 2 MDa Ash1 complex and a 500 kDa Ash2 complex (Papoulas, 1998).
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