moira

Targets of Activity

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

Osa functions as a component of Brm chromatin-remodeling complexes and might be acting through the Brm complex to repress Wg target genes. Other components of the Brm complex were therefore tested for genetic interactions with the wg pathway. Blocking Wg signaling at the wing margin by expressing UAS-Sgg* with vg-Gal4 causes a reduction in wing growth and a loss of the wing margin. These phenotypes are strongly enhanced in flies heterozygous for wg or in those that coexpress UAS-Osa; they are suppressed in flies heterozygous for axin (a negative regulator of Wg signaling or osa. The effects of UAS-Sgg* expression are also suppressed by the loss of one copy of brm or moira (mor), which encodes an essential component of the Brm complex, or by coexpression of a dominant negative form of Brm (DN-Brm). In contrast, two other trithorax group genes [trithorax (trx) and absent, small, or homeotic discs 2 (ash2)] that encode components of other nuclear complexes thought to regulate chromatin structure, failed to modify the UAS-Sgg* phenotype (Collins, 2000).

This demonstrates that there is a specific genetic interaction between the wg pathway and components of Brm complexes and suggests that these complexes are required for the repression of Wg target genes. Indeed, the wg-dependent gene nub is ectopically expressed in wing discs that contain large clones of cells mutant for brm or mor or that expressed DN-Brm in the dorsal compartment. Furthermore, the loss of nub expression caused by expression of UAS-Osa with ap-GAL4 is rescued by coexpression of DN-Brm, indicating that Brm activity is required for the repression of Wg target genes by Osa. The Wg-dependent UbxB-lacZ reporter is also de-repressed in embryos that express DN-Brm, and coexpression of DN-Brm can rescue the loss of UbxB-lacZ expression caused by DN-Pan. These results suggest that Osa acts through the Brm chromatin-remodeling complex to prevent the expression of Wg target genes (Collins, 2000).

Protein Interactions

In vitro, Mor can bind to itself, via the leucine zipper domain, and it interacts with Brahma (BRM), a SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The association between Mor and Brm may be mediated by 507 amino acids in Brm that include domain II. Deletion of this region is known to cause a decrease in the size of the Brm complex, presumably due to the loss of one or several subunits. The SAND domain of Mor may play a role in the association of Mor with domain II and adjacent residues of Brm. The demonstration that Mor is able to self-associate raises the possibility that it is present in two copies in each complex, similar to BAF170 and BAF155, which are both present in each human complex. these results support a dimer-like model for the structure of the SWI-SNF complex, with duplication of some or all subunits. Such a model has been proposed because the overall molecular mass of the complex is much greater than the sum of its individual components (Crosby, 1999).

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. Western blotting has identified BAP45 as Snr1. The BAP155 protein is highly related to the BRG1/hBRM associated factors (BAFs) BAF155 and BAF170, and the yeast SWI3 and RSC8 proteins. Common to all of these proteins are three domains of unknown function: regions I, II and III. The 440 residues between the N terminus of BAP155 and domain I are highly conserved in the human BAF155 and BAF170 proteins (39% and 34% identity, respectively), but not in the yeast SWI3 and RSC8 proteins. SWI3 and RSC8 also lack the proline-rich domains immediately C-terminal to domain III that are present in BAP155 and its human counterparts. The BAP60 protein is highly related throughout its length to BAF60a, BAF60b and BAF60c, the human homologs of the yeast SWP73 and RSC6 proteins. BAP60 is most closely related to BAF60a (72% identity), which is consistent with the characterization of BAF60c as a potentially tissue-specific subunit and with the identification of BAF60b as a component of a variant 500 kDa complex in mammals. BAP60 is equally related to both yeast SWP73 and RSC6 proteins (approximately 16%-28% identity); however, the yeast proteins contain two relatively large insertions within the approximately 370 amino acid segment that is conserved in their Drosophila and human relatives. Thus the BRM complex contains four subunits (BRM, BAP155, BAP60 and BAP45/SNR1) that are conserved in the human BRG1 and hBRM complexes and in both the yeast SWI/SNF and RSC complexes (Papoulas, 1998 and references).

In addition to counterparts of the yeast SWI/SNF and RSC subunits, the BRM complex contains a polypeptide unique to higher eukaryotes. Peptide sequences obtained for BAP111 matched the translation of a Drosophila EST, LD13023, which encodes an HMG domain protein. This EST overlaps another Drosophila EST (LD03794) that was previously identified by Wang (1998) in a search for sequences related to an HMG domain-containing subunit of the human BRG1 and hBRM complexes, BAF57. Like BAF57, the Drosophila BAP111 protein contains the conserved proline, tyrosine and lysine residues characteristic of HMG-domain proteins that recognize structured DNA without sequence specificity (Wang, 1998). The BAP111 subunit of the BRM complex is thus conserved in higher eukaryotes but is absent from the yeast SWI/SNF and RSC complexes (Papoulas, 1998).

Identification of the remaining three BAPs reveals proteins not previously reported to be subunits of chromatin remodeling complexes. Peptides from BAP55 match the translation of a Drosophila EST that appears to encode a novel actin-related protein. Actin related proteins (Arps) are a functionally diverse group of proteins that share 17%-64% sequence identity with actin. The translation of sequence obtained from both ends of the BAP55 cDNA reveals 38% identity with actin over a total of 239 amino acid residues (comprising the 157 N-terminal and 84 C-terminal residues of BAP55) suggesting it is one of the more divergent Arps. These regions of BAP55 are even less related to other known Arps. Because antibodies to BAP55 do not exist, it could not be determined whether BAP55 is a nuclear protein and a bona fide subunit of the BRM complex by immunoprecipitation. However, it is intriguing that some of the most divergent Arps identified to date are nuclear proteins with reported roles in transcription and chromatin structure (Papoulas, 1998).

Two peptides identify BAP74 as the HSP70 cognate HSC4 (the product of the Hsc70-4 gene). HSC4 is a constitutive (non-heat inducible) chaperone protein. Peptide sequences from BAP47 match conserved regions of the non-muscle actins ACT1 and ACT2 (products of the Act42A and Act5C genes). Due to the extreme abundance of actin and HSC4 in the embryo, immunoprecipitation experiments were unable to demonstrate a clear association of these proteins with the BRM complex. Consistent with these findings, both actin and an actin-related protein have recently been identified as subunits of the human hBRM and BRG1 complexes (K. Zhao, W. Wang, O. Rando, Y. Xue and G. Crabtree, personal communication to Papoulas, 1998).

The yeast SWI/SNF complex has been reported to associate with the RNA polymerase II holoenzyme. This claim has been challenged and conflicting reports have emerged regarding the mammalian hBRM and BRG1 complexes and Polymerase II. None of the seven BAPs are PolII subunits; antibodies against the second largest subunit of PolII fail to detect any antigen in purified BRM complex by western blotting. Therefore PolII of Drosophila does not appear to be stably associated with the BRM protein in Drosophila embryo extracts (Papoulas, 1998 and references).

None of the identified BAPs are known trx-G proteins. Since many of the trx-G genes have not yet been cloned, might one or some encode any of the newly identified subunits of the Brm complex? Using a combination of hybridization to a filter containing mapped P1 clones (9216 clones with an average of 83 kb of genomic DNA per clone) and in situ hybridization to polytene chromosomes, a single map location for each of the previously unmapped BAPs was found. The P1 clone number and cytological position for each of these BAPs is as follows: BAP155, P1# DS08140, map location 88E9-F2; BAP111, P1# DS00459, map location 8C9-13; BAP60, P1# DS03747, map location 11D5-10; and BAP55, P1# DS01093, map location 54A2-B. The P1 clone hybridizing to BAP155 is reported to map to 88E9-F2, very close to the location assigned to the trx-G gene moira (mor). None of the other BAPs map near known trx-G genes. Among all of the trx-G genes analyzed (including dev, kis, mor, osa, skd, sls, ash1, ash2, trx, Trl, urd, snr1 and vtd) only moira was found to enhance a dominant negative brahma mutation. Thus, with the possible exception of mor, the sequence and chromosomal map location of the BAPs does not correspond to previously identified trx-G genes. Only mor genetically interacts with brahma. It is therefore concluded that the majority of trx-G proteins are not prominent subunits of the Brm complex and their functions are not essential for Brm function in vivo (Papoulas, 1998).

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. The requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes, have been dissected in this study. Reconstituted transcription reactions have established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex has revealed a number of novel subunits. Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and Osa. The leucine zipper of Zeste mediates binding to Mor. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting (Kal, 2000).

The BRM complex is not required for promoter binding by Zeste, suggesting that it functions at a later step during the transcription cycle. Restructuring of the local chromatin environment by the recruited BRM complex may allow for the subsequent recruitment of other coactivators and the transcription machinery. In yeast cells, such an ordered recruitment has been observed at the HO promoter. The notion that Zeste recruits the BRM complex is further supported by the catalytic amounts of BRM complex needed to mediate Zeste-directed transcription. A BRM-to-nucleosome molar ratio of less than 1:50 is estimated. Recently, direct recruitment of the yeast SWI/SNF complex by an acidic activation domain has been reported. Because Zeste contacts the BRM complex through different protein motives, it will be of interest to determine what subunits of the yeast SWI/SNF complex are contacted by acidic activators. This may establish whether different activators target distinct subunits in SWI/SNF-type remodeling complexes (Kal, 2000 and references therein).

Can the recruitment mechanism described here be generalized? Although the majority of PcG and trxG proteins associate with specific chromosomal sites, they do not appear to bind DNA directly. Response elements for PcG and trxG proteins (PREs) are poorly defined sequences of several hundred base pairs. In addition to Zeste, there are a few candidate sequence-specific tethering factors such as Pleiohomeotic and GAGA. Thus far it has been impossible to reduce PREs to a number of simple sequence motives, therefore it is likely that there will be additional DNA-binding proteins that function as anchors for PcG and trxG proteins (Kal, 2000 and references therein).

Zeste and BRM both belong to the trxG proteins that have been identified as transregulators of homeotic gene function in Drosophila. The majority of Brahma-associated proteins (BAPs) are not encoded by trxG genes and several other trxG proteins have been found to be part of separate protein complexes. Thus, it has been unclear whether distinct trxG proteins may cooperate in a single biochemical pathway. This study now establishes a direct physical interaction between four distinct trxG proteins during transcriptional activation. Previous analysis of osa and mor has revealed a strong genetic interaction of these genes with brm. MOR and OSA are shown to be integral constituents of the BRM complex that are directly contacted by Zeste. Genetic studies have indicated that MOR, OSA, BRM, and Zeste share at least some target genes. These results now provide a biochemical basis for the functional relationship between these trxG proteins (Kal, 2000 and references therein).

Purification of the BRM complex and stringent coimmunoprecipitation experiments have suggested the presence of two novel core BAPs in addition to OSA: BAP170 and BAP26. Moreover, it appears that there are several less tightly associated proteins. The idea is favored that the interaction of the majority of these proteins with the core BRM complex is specific, because they copurify over several columns and remain associated during selective coimmunoprecipitation. Moreover, Zeste specifically interacts with the p400 complex component, supporting the notion that its association with the BRM complex is functional (Kal, 2000).

Do distinct remodeling factors perform different functions or are they redundant? A number of recent studies shed light on the basic mechanisms by which SWI/SNF and ISWI remodelers catalyze nucleosome mobilization. However, their potential roles as regulators of transcription are still poorly understood. Presented here is a clear example of functional differentiation among chromatin remodelers. The BRM complex is an essential coactivator for Zeste, whereas the ISWI family members are not required. Reversibly, at least some ISWI remodeling factors appear to be more efficient at ordering nonperiodic nucleosomal arrays than the BRM complex. Thus, this side-by-side comparison of distinct endogenous Drosophila remodeling factors shows that each performs distinct specialized functions (Kal, 2000).

The SWI/SNF-type remodelers appear to function in a highly selective manner. For example, the human SWI/SNF-related chromatin-remodeling complex, E-RC1 is required for the activation of the beta-globin gene by the activator EKLF but does not work with another transcription factor, TFE3. Moreover, it is pertinent to note that the yeast and Drosophila SWI/SNF family members were first identified by genetic screens for gene-specific regulators. Thus, studies in yeast, mammals, and Drosophila all point to an integral and essential role for SWI/SNF remodelers in gene-specific transcriptional regulation. Although the ISWI remodelers are not required for Zeste function, they have been implicated in transcriptional activation by other regulators such as GAL4-VP16. Several lines of evidence suggest that ISWI remodelers may act by a mechanism that is fundamentally distinct from that of the SWI/SNF family complexes. For example, unlike NURF, SWI/SNF does not seem to require the histone tails for remodeling. Moreover, studies on NURF suggest that it remodels chromatin in a transient nonspecific manner, creating an opportunity for transcriptional activators to bind DNA. Such a mode of action does not involve the direct physical interactions between remodeler and activator described in this study for the BRM complex. In conclusion, all available evidence points to an extensive functional specialization of ATP-dependent chromatin remodeling factors. An attractive possibility is that different genes require the action of distinct subsets of remodeling complexes, histone acetyl transferases, and other coactivators. Such a combinatorial arrangement would vastly expand a cell's potential for precise and coordinated regulation of individual genes (Kal, 2000).

DEVELOPMENTAL BIOLOGY

Embryonic

moira is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. Protein is ubiquitous early in development and highly expressed in the central nervous system and the gut of older embryos. Since the protein is expressed in all somatic nuclei of syncytial and cellularized blastoderm stage embryos, it is likely that the mor gene is maternally expressed. At germ band lengthening, higher levels of Mor are seen in the developing gut (the invaginating endoderm of the posterior midgut and the stomodeum) and the ventral nerve cord. In the latter tissue the nuclear localization of Mor is still apparent. Upon germ band retraction Mor is preferentially enriched in the mid- and hind-guts and in the ventral nerve cord and the brain (Crosby, 1999).

Effects of Mutation or Deletion

Proteins produced by the homeotic genes of the Hox family assign different identities to cells on the anterior/posterior axis. Relatively little is known about the signaling pathways that modulate the activities of these proteins activities or the factors with which they interact to assign specific segmental identities. To identify genes that might encode such functions, a screen was carried out for second site mutations that reduce the viability of animals carrying hypomorphic mutant alleles of the Drosophila homeotic locus, Deformed. Genes mapping to six complementation groups on the third chromosome were isolated as modifiers of Deformed function. Products of two of these genes, sallimus and moira, have been previously proposed as homeotic activators since they suppress the dominant adult phenotype of Polycomb mutants. Mutations in hedgehog, which encodes secreted signaling proteins, were also isolated as Deformed loss-of-function enhancers. hedgehog mutant alleles also suppress the Polycomb phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with Polycomb, indicating that the screen identifies genes that are not general homeotic activators. Two of these genes, cap 'n' collar and defaced, have defects in embryonic head development that are similar to defects seen in loss-of-function Deformed mutants (Harding, 1995).

The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16^ink4a/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. An E2F overexpression phenotype in the Drosophila eye was use to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. Thirty three enhancer mutations of this phenotype were isolated by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma (brm), moira (mor), osa, pointed (pnt), and polycephalon (poc). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21^CIP1 expression, indicating an important role for Polycephalon in cell-cycle control (Staehling-Hampton, 1999).

The molecular basis of the interaction between E2F and a BRM/MOR/OSA chromatin-remodeling complex is not yet clear and a range of possibilities exists. The genetic interaction may result from a direct physical interaction between RBF/E2F complexes and chromatin-remodeling machinery. In support of this idea the human homologs of BRM, hBRM, and BRG1 have been found to physically associate with pRB. This raises the possibility that BRM/MOR/OSA may help E2F/RBF repressor complexes bind to their target sites. This interpretation is supported by experiments from Trouche and co-workers who used transient transfection of mammalian cells to demonstrate that BRG1 can cooperate with pRB to repress E2F-dependent transcription (Trouche, 1997). Consistent with this model, the introduction of two copies of GMR-RBF into a GMR-dE2FdDPp35/+; brm^-/+ background suppresses the enhancement by brm. Thus the effect caused by low levels of brm can be overcome by increasing the dosage of RBF. Additional evidence has been sought that would be predicted by this model; to date, however, these experiments have been inconclusive. BRM lacks the LXCXE motifs found in hBRM and BRG1, which have been suggested to mediate the interaction with pRB. To date no physical interaction between BRM and RBF or between BRM and dE2F has been detected. The interaction between endogenous pRB and hBRM or BRG1 proteins is hard to detect even in mammalian cells, and the failure to find BRM/RBF complexes may simply reflect difficulty in extracting chromatin-associated proteins under conditions that maintain the interaction (Staehling-Hampton, 1999).

An alternative possibility is that the BRM/MOR/OSA chromatin-remodeling complex is an important regulator of the expression of some key E2F-target genes, but this complex does not interact directly with either RBF or E2F. In this case the functional interaction occurs because these proteins converge on overlapping sets of promoters. This model is difficult to test because it is not yet clear which, and how many, E2F target genes are functionally significant. RNR2, one example of an E2F-dependent gene, is expressed normally in embryos mutant for brm, osa, or mor; no change in the expression of RNR2 in GMR-dE2FdDPp35 eye disks heterozygous for brm, osa, or mor alleles could be detected. While RNR2 expression is often used to provide an in vivo readout of E2F activity, experiments suggest that it is not a critical E2F target. The effects of brm, mor, and osa may only be evident at a subset of E2F-regulated promoters and an extensive screen of E2F targets will be necessary to find the appropriate gene (Staehling-Hampton, 1999).

It is possible that E2F and brm act in distinct pathways that influence cell-cycle progression. In this model the activity of a BRM/MOR/OSA-containing complex may have a function that influences the ability of E2F or RBF to control S-phase entry. Several observations have linked BRM-related proteins to cell-cycle control. brm null clones in the adult cuticle often show duplications of bristle structures, suggesting a possible role for brm in proliferation, and mice lacking the BRM homolog SNF2alpha show evidence of increased cell proliferation. Although brm, mor, and osa have no effect on the GMR-p21 phenotype, both brm and mor mutations have been isolated as suppressors of a hypomorphic cyclin E eye phenotype, demonstrating that brm and mor can affect other cell-cycle phenotypes in the eye. Other studies have shown that the activity of hSWI/SNF complexes is itself cell-cycle regulated. Transformation by activated Ras decreases the expression of the murine ortholog of hBRM in mouse fibroblasts, whereas growth arrest leads to an accumulation of protein. Recently, BRG1 and BAF155, a human ortholog of Moira, have been shown to associate with cyclin E and are suggested to be targets for cyclin E-dependent kinases during S-phase entry (Staehling-Hampton, 1999 and references).

During this study it was observed that GMR-dE2FdDP p35/+; brm^-/+ eyes develop necrotic patches that increase in severity with the age of the adult fly. This raised the possibility that brm mutations might enhance the phenotype by promoting E2F-induced apoptosis. However, further experiments have failed to support this hypothesis. brm mutations fail to enhance the GMR-dE2FdDP phenotype, which has elevated levels of apoptosis, or to modify a GMR-rpr phenotype. In addition, brm mutations have no effect on the phenotype of animals in which GMR-rpr and GMR-hid-induced apoptosis is blocked by GMR-p35. No increase in the number of apoptotic cells is detected when GMR-dE2FdDPp35/+; brm^-/+ third instar eye disks are stained with acridine orange (Staehling-Hampton, 1999).

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DNA replication-related element factor or DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, transgenic flies were established in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induces ectopic DNA synthesis in the cells behind the morphogenetic furrow that are normally postmitotic, and abolishes photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype is suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, it was found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppresses and that reduction of Distal-less enhances the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed identification of several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators (Hirose, 2001).

A mosaic genetic screen reveals distinct roles for trithorax and Polycomb group genes in Drosophila eye development

The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).

trithorax group genes were initially identified as suppressors of Polycomb phenotypes and are therefore thought to contribute to the activation of homeotic gene expression. Some members of the group encode components of the Brahma chromatin-remodeling complex, others encode components of the mediator coactivation complex, and still others encode histone methyltransferases. In addition to their distinct biochemical functions, members of the trithorax group act on different sets of target genes during eye development and can also have different effects on the same target genes. Components of the Brahma complex are strongly required for cell growth and/or survival; brm and mor, but not osa, are also absolutely required for photoreceptor differentiation. However, these three genes do not seem to be required for the restricted expression in anterior-posterior domains of the eye disc of the transcription factors examined. In contrast, the mediator complex subunits encoded by skd and kto are not required for cell proliferation, although they are strongly required for photoreceptor differentiation. trx, which encodes a histone methyltransferase, is required primarily for the normal development of marginal regions of the disc. No significant effect on photoreceptor differentiation were seen in clones mutant for kismet¹, which encodes chromodomain proteins, or ash2¹, which encodes a PHD protein. These differences are unlikely to be due to different expression patterns of the trithorax group genes, since Trx, Skd, Kto, and Osa are ubiquitously expressed in the eye disc (Janody, 2004).

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date revised: 15 September 2004

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