Transcriptional Regulation

Transcriptional regulation of the Drosophila moira and osa genes by the DREF pathway

The DNA replication-related element binding factor (DREF) plays an important role in regulation of cell proliferation in Drosophila, binding to DRE and activating transcription of genes carrying this element in their promoter regions. Overexpression of DREF in eye imaginal discs induces a rough eye phenotype in adults, which can be suppressed by half dose reduction of the osa or moira (mor) genes encoding subunits of the BRM complex. This ATP-dependent chromatin remodeling complex is known to control gene expression and the cell cycle. In the 5' flanking regions of the osa and mor genes, DRE and DRE-like sequences exist which contribute to their promoter activities. Expression levels and promoter activities of osa and mor are decreased in DREF knockdown cells and the results in vitro and in cultured cells indicate that transcription of osa and mor is regulated by the DRE/DREF regulatory pathway. In addition, mRNA levels of other BRM complex subunits and a target gene, string/cdc25, were found to be decreased by knockdown of DREF. These results indicate that DREF is involved in regulation of the BRM complex and thereby the cell cycle (Nakamura, 2008).

This study demonstrated that both osa and mor are DREF target genes. Thus osa and mor promoters exhibited decreased activities when carrying mutations in their DREs and after knockdown of DREF in cultured cells. In addition, levels of osa and mor mRNAs were reduced in DREF knockdown cells. Third, DREF can bind to DREs of osa and mor in vitro, and binding of DREF to the genomic regions containing DREs of both genes was observed in cultured cells. These results showed that DRE and DREF are important for osa and mor promoter activation. Promoters having mutations in all DREs of both osa and mor genes, however, still retained some activity. It is therefore possible that another element(s) and/or unknown factor(s) regulated by DREF are involved in osa and mor transcriptional activation. The observed rescue of the DREF-induced rough eye phenotype by a reduction in the osa and moire gene dosage is consistent with the idea that the osa and moire gene transcription is activated by DREF. However,the possibility cannot be excluded that the rescue could also be affected by a mechanism involving protein-protein interactions between DREF and BAP/PBAP at the promoters of cell cycle-regulated genes. Further analyses are necessary to address this point (Nakamura, 2008).

Both osa and mor encode components of the BRM complex, which is a SWI/SNF type ATP-dependent chromatin remodeling complex conserved from yeast to human, with two forms, BAP and PBAP. Osa is a signature subunit of BAP, while PBAP contains Polybromo and BAP170 in its place. Localization patterns of Osa and Polybromo on polytene chromosomes differ, though several sites overlap. Whole-genome expression analysis also demonstrated that BAP and PBAP differentially regulate gene expression. For example, Osa negatively regulates expression of the Wingless-target genes and the achaete/scute gene. Osa, Polybromo and BAP170 are all required for function of BRM complex. It is thought that Osa functions in recruitment of BAP to its target genes. Mor, a subunit common to both BAP and PBAP, is presumed to be essential for complex integrity, since its absence results in degradation of both forms. SRG3, which is a homolog of Mor in mammals, also acts for complex stabilization by protecting against proteasomal degradation. Therefore, Osa and Mor are essential subunits for function and stabilization of BRM complexes and DREF may control integrity of the BRM complex through activating osa and mor gene expression (Nakamura, 2008).

BAP and PBAP share seven subunits, Brm, Mor, Snr1, BAP111, BAP60, BAP55 and Actin. Brm is a catalytic subunit harboring the ATPase domain and it was previously reported that reduction of the brm gene dose suppressed the DREF-induced rough eye phenotype. It was also found the the mRNA level of brm is decreased in DREF knockdown cells. However, DRE-like sequences in the second intron, do not appear to function as regulatory elements, since DREF does not bind to the genomic region containing these sites in vivo. DREF may therefore indirectly control brm gene expression (Nakamura, 2008).

The genes coding for BAP55 and BAP60, common subunits for BAP and PBAP, also contain DRE or DRE-like sequences in their 5' flanking regions and are affected by DREF knockdown. DREF binds to the genomic regions containing their DREs in vivo and it is, therefore, possible that BAP55 and BAP60 are directly regulated by DREF. Furthermore, the PBAP-specific subunit BAP170 carries a DRE in its 5' flanking region. Reduction of mRNA levels of osa, polybromo and BAP170 in DREF knockdown cells also is evidence that DREF contributes to the transcriptional regulation of both BAP and PBAP complexes. Therefore, DREF may regulate expression of genes coding for most subunits for both BAP and PBAP complexes and influence expression of many genes through chromatin remodeling (Nakamura, 2008).

It has been reported that OSA-containing BAP complexes are necessary for G2/M progression through stg promoter activation while PBAP complexes are not. stg encodes a CDC25 phosphatase, which is required for G2/M progression. It is well known that DREF predominantly regulates the transcription of DNA replication-related genes. Reduced stg mRNA has been reported in DREF-eliminated cells and this study also observed reduction of stg mRNA levels in DREF knockdown cells, as with brm, osa and mor. In addition to regulation of S phase entry, DREF thus appears to play an important role in G2/M transition by activating the BAP complex to promote cell cycling. Two DRE-like sequences were found in the stg gene upstream region, -219 to -212 (5'-aATCGATg) and -591 to -584 (5'-TATCGATt). Therefore, DREF could regulate stg gene expression directly via binding to DRE-like and/or indirectly via activation of genes coding for BAP complexes. Further analysis is necessary to distinguish these possibilities (Nakamura, 2008).

BRM complexes are thought to inhibit S phase entry and mutations of brm, osa and mor suppress the rough eye phenotype induced by E2F/DP/p35 overexpression. The rough eye phenotype of a cyclin E hypomorphic mutant was also suppressed by BRM complex mutation through increase in the S phase. Therefore, BRM complexes appear to negatively regulate S phase entry, while DREF activates E2F gene transcription and promotes G1/S progression. Although osa is ubiquitously expressed in eye imaginal discs, it is most intensely expressed anterior to the morphogenic furrow where cells enter the G1 phase. Similarly, DREF is strongly expressed in this region. It is conceivable that DREF simultaneously activates both positive and negative regulators of G1/S progression. This kind of regulation may be necessary for fine tuning of cell cycle progression to inhibit excess S phase induction (Nakamura, 2008).

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).

Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes

The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. This was addressed by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. These studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie (Oh, 2013).



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 p16ink4a/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 p21CIP1 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).

Osa, a subunit of the BAP chromatin-remodelling complex, participates in the regulation of gene expression in response to EGFR signalling in the Drosophila wing

Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the Bramha associated protein (BAP) complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. This work analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Mor (Moira) and Brahma, also share these functions of Osa. Focus was placed on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. It is suggested that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling (Terriente-Félix, 2009).

Chromatin structure is critical to modulate gene expression during development, and is affected by a variety of alterations such as histone modification, DNA methylation and changes in conformation. Proteins related to Drosophila Brm, such as yeast SNF2 modify chromatin in an ATP-dependent manner, causing repositioning of nucleosomes along the DNA and re-distribution of histone proteins between nucleosomes. The SWI/SNF complexes are conserved in all eukaryotes, and display specific interactions with distinct transcription factors to regulate different subsets of genes. There are several examples where sequence-specific transcription factors interact specifically with SWI/SNF complexes. For example, the ATPase BRG1 binds Zn-finger proteins and hBRM interacts specifically with CBF-1/Su(H), which recruits hBRM to Notch target promoters such as those of HES1 and HES5 (Terriente-Félix, 2009).

A key aspect in the analysis of Brm function is the identification of targets accounting for the functions of the complex. A necessary step in this analysis is the description of its functional requirements using genetic approaches; which helps to identify the specific processes affected by loss of BAP function. The current data indicate that Osa is required during wing disc development for cell viability, cell proliferation, and for the formation of wing veins and the wing margin. Interestingly, increased expression of Osa in the wing also causes phenotypes related to wing growth and patterning, such as reduced wing size, ectopic sensory organs and hairs and the formation of extra vein tissue in most interveins. This analysis focused mostly on Osa, and this raises the question of whether its requirement reflects the function of the BAP complex. This is the most likely scenario, because the preliminary analysis of other BAP members, such as Snr1, Bap55, Mor and Brm uncovers similar phenotypes in the wing. Thus, lowering Snr1, Bap55 or Mor levels reduces wing size, disrupts the wing epithelium and causes the differentiation of ectopic sensory organs and hairs. These wings also display loss of veins, and in general the overall phenotypes are similar to those of loss of Osa. The phenotype of iRNA expression directed against brm is much milder, perhaps due to a lower efficiency of this construct, but still these wings show a loss of veins phenotype. The reduction of Bap170, a member of the PBAP complex, causes the formation of ectopic veins, which is the opposite phenotype to loss of function in osa and in other members that are present in both the BAP and PBAP complexes. Thus, although Brm is the catalytic subunit in both BAP and PBAP, these complexes could act in opposite manners on the same target genes at least during wing vein formation (Terriente-Félix, 2009).

Some Osa requirements can be explained by modifications in the transcriptional response to the activity of the Wg signalling pathway and by effects on wg expression. The function of Wg is required for the formation of the wing margin, including the development of sensory organs and veins along the anterior wing margin. In the absence of Wg signalling the wing margin does not form, and when Wg signalling is inappropriately activated ectopic sensory organs and hairs differentiate throughout the wing blade. In addition to affecting the response to Wg signalling, Osa is also required for the expression of wg along the dorso-ventral boundary. This requirement might be related to Notch signalling in these cells, and explains why the remnants of wing tissue formed in osa mutant wings do not form the wing margin or ectopic sensory organs (Terriente-Félix, 2009).

This study focused on the characterisation of Osa during the formation of the longitudinal wing veins. This process is independent of Wg signalling, and requires the activities of the Notch, Dpp and EGFR signalling pathways. Osa is needed for the expression of bs in the interveins, because bs is not expressed in cells mutant for osa. The regulation of bs expression involves the activity of Ash2 and the function of the Hh and Dpp pathways. It is suggested that Osa participates in the activation of bs facilitating the availability of its regulatory regions to these activators. This aspect of Osa function does not explain the phenotype of loss of veins characteristic of osa mutant cells, because the loss of Bs expression is normally associated with the differentiation of ectopic veins. The only context where bs mutant cells differentiate as interveins is when the activity of the EGFR pathway is reduced. Therefore, it is suggested that loss of bs expression is accompanied in osa mutant cells by a failure in the response to EGFR activity, leading to the differentiation of intervein tissue. Interestingly, the expression of bs is also severely reduced when Osa is present at higher than normal levels, and in this case loss of Bs is accompanied, as expected, by the formation of ectopic veins. The effects of increased Osa on bs expression can also be explained if Osa facilitates EGFR activity, because this pathway mediates the repression of bs in the proveins. In both cases, the common aspect mediated by Osa might be to regulate bs expression in collaboration with its transcriptional activators and repressors (Terriente-Félix, 2009).

Because the failure of osa mutant cells to differentiate the veins is not due to changes in bs expression, nor to changes in the expression of provein genes such as kni and caup, the search for Osa candidate targets was narrowed to the EGFR pathway. Several results suggest a close relationship between Osa and EGFR signalling in the wing. First, the phenotypes of changing osa expression in the veins are very similar to those resulting from the same manipulation in EGFR activity. Thus, a reduction in any core component of the EGFR pathway eliminates the veins, whereas the increase in EGFR signalling activity causes the formation of extra veins in intervein territories. Second, genetic interactions were observed between osa and several components of the EGFR pathway compatible with a function of Osa promoting EGFR activity in the veins. Finally, the extra veins caused by excess of Osa are suppressed when the activity of EGFR is reduced, indicating that Osa cannot substitute for EGFR activity. The changes in vein and intervein expression patterns are already detected in the wing disc, before other signalling pathways, such as Dpp, act to promote vein formation. Taken together, these observations suggest that Osa facilitates the response to EGFR activity in the wing disc, but cannot promote the transcription of EGFR targets in the absence of EGFR signalling (Terriente-Félix, 2009).

The changes in the expression of EGFR target genes observed in osa mutant cells or in osa gain-of-function experiments are compatible with a direct function of Osa/BAP is the transcriptional regulation of EGFR targets such as Dl, rho and aos. How Osa and the BAP complex are targeted to specific genomic regions is not entirely clear, although it is likely that sequence-specific transcription factors are involved in this process. Transcription in response to EGFR signalling is mediated by proteins belonging to the ETS family, such as Pointed-P2, Pointed-P1 and Yan in Drosophila. However, these genes are not required during wing vein formation, suggesting that other ETS proteins or uncharacterised transcription factors bring about interactions between the regulatory regions of EGFR target genes and the BAP complex (Terriente-Félix, 2009).

It is unlikely that Osa participates in any step of the EGFR pathway previous to the transcription of its target genes. It was noticed, however, that the expression of dP-ERK, a direct read-out of the pathway activity, is also affected in osa mutant cells. Thus, these cells frequently fail to express normal levels of dP-ERK, a result indicating that EGFR activity is reduced. The most likely explanation for this observation is that, in the wing, the EGFR pathway is engaged in a positive feedback loop mediated by the activation of rho expression, which maintains EGFR activity in cells where it has already been activated. Thus, loss of osa leads to a failure to express rho and subsequently to a reduction in the activity of the pathway detected as a loss of dP-ERK expression. There is one experimental situation in which Osa function appears to be dispensable for the expression of EGFR target genes. Thus, when a constitutive active form of Ras, RasV12, is driven in the wing, the augmented expression of Dl and aos, and the accumulation of dP-ERK are not affected by a reduction in Osa levels. It is possible that in this situation of strong and constitutive activity of the pathway, the possible modifications to chromatin structure brought about by Osa/BAP on EGFR target genes are not necessary, perhaps because at this level of EGFR activation the transcriptional repressors antagonising EGFR target gene transcription, such as Cic and Gro, are inactivated by the pathway, and this might make dispensable the function of Osa (Terriente-Félix, 2009).

It is not entirely clear to what extent the link observed between BAP function and EGFR signalling during wing disc development is conserved in other developmental systems and in other organisms. Some phenotypes of osa and brm alleles described in the eye disc, such as the loss of photoreceptor cells, are also observed upon a reduction in EGFR activity. Similarly, the loss of distal growth in the legs is also characteristic of reduced EGFR activity. These data are indicative of a general requirement for Osa in the expression of EGFR target genes at least in imaginal discs. The genetic approach that was used identifies transcription downstream of EGFR signalling as a relevant in vivo function of BAP complexes. Subsequent biochemical analysis should determine whether the functional interactions that were observed are mediated by direct binding of BAP to the regulatory regions of bs and other EGFR target genes (Terriente-Félix, 2009).

SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster

The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA. This study investigated whether the function of Brm in pre-mRNA processing in Drosophila is mediated by Brm alone or by the SWI/SNF complex. The effects of depleting individual SWI/SNF subunits on pre-mRNA processing was examined throughout the genome, and a subset of transcripts was identified that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. Brm was also depleted in larvae, and it was shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo. This study has shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. These results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes (Waldholm, 2011).

Previous studies have shown that Brm influences the alternative processing of a subset of pre-mRNAs in human and insect cell lines (Batsche, 2006; Ito, 2008; Tyagi, 2009) but the mechanisms responsible for such regulation are not known. In these studies, a functional link was found between the levels of Brm and the splicing outcome was established after experimental alteration of the Brm levels in cultured cells, either by over-expression or by RNAi-mediated depletion. These studies focused on the Brm subunit. This study has now extended the previous studies and asked whether depletion of other SWI/SNF subunits also results in alterations of pre-mRNA processing. In addition to Brm, this study disrupted Mor, Snr1, PB, Bap170 and Osa in Drosophila S2 cells. Also, microarray data was mined, looking for genes whose relative abundances between alternative transcripts are changed by the RNAi treatment in a different manner than the abundances in mock-treated samples. The number of genes affected was low, but many of the detected events could be validated. This small collection of validated genes is valuable for future mechanistic studies (Waldholm, 2011).

The reasons for the low number of genes affected could be partly technical. First, the data used was obtained from expression arrays that do not cover all the splicing variants of the transcriptome of Drosophila. Second, the variances in the datasets were relatively high and, for this reason, attempts were made to avoid false positives by establishing stringent criteria and discarding genes that did not show consistent results in the replicates. In spite of these limitations, a total of 45 genes were identified for which the pre-mRNA processing levels changed after depletion of SWI/SNF subunits. Depletion of different SWI/SNF subunits affected different genes with a statistically significant overlap, in particular for the core subunits of the SWI/SNF complex. Indeed, a group of ten genes were identified that, according to the microarray data, were affected by depletion of at least two different core subunits. In summary, these results show that depletion of other core subunits apart from Brm influences pre-mRNA processing. This conclusion agrees with observations in human cells, where Brm modulates the splicing of the TERT transcripts in concert with the mRNA-binding protein p54(nrb) (Ito, 2008). In the same study, it was shown that p54(nrb) and core subunits of the SWI/SNF complex interact physically (Waldholm, 2011).

This study has analyzed the decay of the transcripts affected by depletion of SWI/SNF subunits and differential stability can be ruled out as a major cause for the differences observed in the relative abundances of alternative transcripts. Using ChIP, it was also shown that Brm, Snr1 and Mor are asociated with the genes affected. Altogether, these observations support the conclusion that the mechanism by which SWI/SNF affects pre-mRNA processing is direct and cotranscriptional (Waldholm, 2011).

Alternative splicing and polyadenylation are major sources of transcript diversity and proteomic diversity in higher eukaryotes. Complex regulatory networks determine the premRNA processing outcome and play critical roles in differentiation and development. Key elements in such regulatory networks are the splicing and polyadenylation factors that influence the choice of alternative processing sites by binding to cis-acting elements, either enhancers or silencers, in the pre-mRNAs. Recent research has revealed that, in addition to the RNA sequence itself, the chromatin environment and the transcription machinery contribute to the recruitment of regulatory factors to their target transcripts during transcription. Genome-wide studies have shown that certain histone modifications are non-randomly distributed in exons and introns, and that nucleosomes are enriched in exonic sequences. The functional significance of these observations is not fully understood, but there are examples of adaptor proteins that bind both to splicing factors and to specific histone modifications, and such adaptors may play important roles in the targeting of regulatory factors to the pre-mRNA. Another determinant of the splicing outcome is the elongation rate of RNAP II. A reduction of the RNAP II elongation rate at specific positions along the gene can facilitate the assembly of the splicing machinery at weak splice sites and promote the inclusion of proximal exons. hBrm regulates the alternative splicing of the CD44 pre-mRNA in human cells by decreasing the elongation rate of RNAP II and inducing the accumulation of the enzyme at specific positions in the gene. In the case of the CD44 gene, Brm favors the usage of proximal processing sites. This study has shown that in Drosophila the SWI/SNF complex regulates the processing of a subset of pre-mRNAs through somehow different mechanisms. In some of the cases that this study has analyzed, depletion of SWI/SNF promotes the use of a proximal splice site (for instance, the up-regulation of the lola-RA transcript), which cannot be explained by the same mechanisms that act on the human CD44 gene. Several alternative mechanisms can be envisioned. In one scenario, SWI/SNF either decreases or increases the transcription rate, depending on the genomic context and on the presence of specific regulators. Alternatively, SWI/SNF could act by a mechanism that is independent from the transcription kinetics. It was previously shown that a fraction of SWI/SNF is associated with nascent transcripts, while other studies have shown that Brm and specific mRNA-binding proteins interact. It is tempting to speculate that SWI/SNF plays a more direct role in pre-mRNA processing, possibly by modulating the recruitment and/or assembly of splicing or polyadenylation factors (Waldholm, 2011).

Previous research on the role of Brm in pre-mRNA processing was carried out in cultured cells. This study has now depleted Brm in larvae and detected changes in pre-mRNA processing in vivo. Depletion of Brm had no significant effect on two of the four genes analyzed, CG3884 and mod(mdg4). Depletion of Brm in vivo affected lola and Gpdh, in contrast, in a similar manner to its effect in S2 cells. It is important to point out that this study analyzed RNA extracted from total larvae, not from individual organs, which might have occluded tissue-specific effects. Indeed, the gene expression data in FlyAtlas ( shows that the expressions of the analyzed genes vary among organs and throughout development. Analyzing total larvae gives an average of the effects in the entire organism, which might not reflect the physiological regulation of the target genes in any specific tissue. However, the fact that Brm depletion affects the processing of the lola and Gpdh transcripts in larvae shows that the reported effect of SWI/SNF on pre-mRNA processing is not an artefact that occurs only in cultured cells (Waldholm, 2011).

The lola and Gpdh genes are structurally very different. Gpdh is a relatively short gene with three alternative mRNAs that encode nearly identical proteins. The alternative processing of the Gpdh pre-mRNA determines the sequence of the 3' UTRs, which can have a profound impact on the stability of the transcripts, their regulation by microRNAs and their translational properties. The lola gene, in contrast, is very long with at least 26 different transcripts that code for a plethora of protein isoforms characterized by different types of DNA-binding motifs. Therefore, in vivo regulation of lola by SWI/SNF affects the abundances of protein isoforms with different biological activities (Waldholm, 2011).

This study has shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts, both in S2 cells and in larvae. The results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes (Waldholm, 2011).


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moira: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 April 2011

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