The spatial and temporal patterns of snr1 expression are similar to those of brm. The highest level of mRNA accumulation for both genes occurs in unfertilized eggs and early embryos, indicating a maternal contribution of both mRNAs. The level of mRNA decreases steadily through embryogenesis until mRNA is undetectable. A low level of mRNA is present in larval and pupal stages but little is found in adult males. Early expression is ubiquitous, but late expression is limited to the central nervous system (ventral cord), and brain (Elfring, 1994).

Unlike BRM mRNA, Brm protein is present at all stages of development, as revealed by Western blotting. Brm protein is expressed at relatively high levels throughout embryogenesis and in pupae; lower amounts of Brm are present in larvae and adult flies. The level of Brm protein was investigated in developing embryos by quantitative Western blotting using a GST-Brm fusion protein as a standard. Approximately equivalent immunoreactivity is observed with 2 ng of purified fusion protein and protein extracted from 3 to 6-hr embryos. Since ~6,000 nuclei are present at this stage of development, it is estimated that there are at least 100,000 molecules of Brm protein per nucleus at its peak stage of expression. This level of expression corresponds to approximately one molecule of Brm protein per 20 nucleosomes, contrasting sharply with the relatively low abundance of SWI2/SNF2 in yeast cells (approximately several hundred molecules per nucleus). The spatial expression of Brm protein was examined by immunostaining whole-mount preparations of embryos and larvae. Brm protein is present at similar levels in nuclei throughout the early embryo. The Brm protein continues to be expressed ubiquitously during the remainder of embryogenesis, although its levels are somewhat enriched in the ventral nerve cord and brain in late embryos. In late third instar larvae, Brm protein is expressed at relatively uniform levels in nuclei of the imaginal discs and other diploid and polytene tissues, including the polytene nuclei of the salivary gland. Thus, in contrast to the previously reported patterns of BRM mRNA expression, the Brm protein is ubiquitously expressed throughout the developing organism (Elfring, 1998).


The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).

To clarify the functional relationship between Kis-L and other chromatin-remodeling factors, their distributions on polytene chromosomes were compared. The distributions of Kis-L and Brm were compared, since previous studies have suggested that the two proteins have similar functions. For example, brm and kis were both identified in genetic screens for dominant suppressors of Pc and mutations in the two genes cause similar homeotic transformations. In addition, Brm plays an extremely general role in transcription by Pol II and, like Kis-L, is associated with almost all transcriptionally active regions of polytene chromosomes. Consistent with a close functional relationship between the two proteins, it was found that the distributions of Brm and Kis-L on polytene chromosomes are virtually identical. In addition, the relative levels of the two proteins do not vary from site to site. The striking similarities between the chromosomal distributions of Brm and Kis-L strongly suggest that the functions of the two trxG proteins are intimately related (Srinivasan, 2005).

Effects of Mutation or Deletion

The brahma gene is required for the activation of multiple homeotic genes in Drosophila. Loss-of-function brm mutations suppress mutations in Polycomb, a repressor of homeotic genes, and cause developmental defects similar to those arising from insufficient expression of the homeotic genes of the Antennapedia and bithorax complexes (Tamkun, 1992). Surviving mutants have a reduced number of sexcomb teeth, held out wings, loss of humeral bristles and patches of lightly pigmented cuticle in the fifth and sixth tergite of males. These are all abnormalities observed in loss-of-function mutations of ANTP-C and BX-C genes (Brizuela, 1994).

The absent, small or homeotic discs1 gene (ash1) is one of the trithorax complex genes. Recessive loss of function mutations in ash1 cause homeotic transformations of imaginal disc-derived tissue that resemble phenotypes caused by partial loss or gain of function mutations in genes of the Antennapedia and bithorax complexes. Mutations in the gene brahma, itself a member of the trithorax complex, interact with mutations in ash1 such that non-lethal ash1 +/+ brm double heterozygotes have a high penetrance of homeotic transformations in specific imaginal disc- and histoblast -derived tissues (Tripoulas, 1994).

Both maternal and zygotic functions of brahma are required during embryogenesis. Removal of the maternal contribution results in early embryonic defects. In addition, the severe abnormalities caused by loss of maternal brahma expression show that the homeotic genes are not the only targets for brahma activation. brahma also interacts with hairy and hedgehog, two transcription factors involved in gene activation and silencing. The complex pattern of interallelic complementation for the 21 brahma alleles suggests that Brahma may act as a multimer (Brizuela, 1994).

The snr1 gene is essential and genetically interacts with brm and trithorax, suggesting cooperation in regulating homeotic gene transcription (Dingwall, 1995).

The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. The osa gene was first identified as a trxG gene in the same genetic screens that identified brm (Kennison, 1988). osa turns out to code for the same transcript as eyelid. Regulation of Antennapedia by Brm and Osa proteins requires sequences 5' to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The Osa protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila Dead ringer proteins. It is proposed that the Osa protein may target the BRM complex to Antennapedia and other regulated genes (Vázquez, 1999).

osa and brm were first identified as suppressors of both the antenna to leg transformation caused by the Nasobemia (Ns) allele of Antp and the extra sex combs phenotype caused by derepression of Sex combs reduced (Scr) in Polycomb (Pc) mutants (Kennison, 1988). While examining genetic interactions among trxG mutations, it was noted that flies heterozygous for both brm and osa mutations have a held-out phenotype rarely seen in flies heterozygous for either mutation alone. The expressivity of the held-out wings phenotype is more severe in combinations of brm with some point mutations in osa than it is with the osa deficiency, suggesting that the osa point mutations make altered proteins that still bind to something in competition with wild-type Osa proteins, but then fail to function. Increasing the dosage of wild-type brm reduces the held-out wings phenotype, as expected (Vázquez, 1999).

The held-out wings phenotype is not rare in Drosophila. It is caused by mutations in many other genes, including dpp. This phenotype was also observed in flies trans-heterozygous for partially complementing brm alleles. Nevertheless, the interaction between brm and osa alleles is unusual because it results from the failure of complementation between mutations in two different genes (non-allelic non-complementation). Although a few other trxG mutations have been shown to interact in double heterozygotes, the penetrance in every other case is far less than that observed for the brm/osa interactions. In fact, the majority of trxG mutations show little if any interaction in double heterozygotes. brm interacts with the trxG genes trx and ash1 to cause partial transformation of the fifth abdominal segment to fourth, and metathorax to mesothorax, but these flies do not hold their wings out at any significantly higher frequency. The basis for the held-out wings phenotype in the brm/osa transheterozygotes was investigated. The Antp gene has two alternative promoters, P1 and P2. Genetic studies have shown that the functions of both promoters are essential. Two mutations that inactivate only the P2 promoter have been described. Flies heterozygous for the P2-specific mutations and the chromosome aberrations that remove P1 function were examined. All combinations appear as wild type, except flies carrying either one of two very specific Antp mutations, which produce chromosome aberrations that remove P1 function in combination with the P2- specific mutations. Many of these flies have held-out wings phenotype indistinguishable from the held-out wings phenotype of the brm/osa transheterozygotes. It is suggested that disruption of P2 promoter activity can result in a held-out wings phenotype. Moreover, when a brm mutation is introduced, there is a significant increase in the penetrance of the held-out wings phenotype. These results strongly suggest that brm is one of the factors required for normal expression of the P2 promoter to prevent the held-out wings phenotype (Vázquez, 1999).

That both brm and osa are required for activation of the Antp P2 promoter is also suggested by their interaction with the Antp Ns mutation. The Antp Ns mutant chromosome has a large insertion (including a second copy of part of the P2 promoter) upstream of the P2 promoter. This insertion derepresses the P2 promoter and causes the antennae to differentiate leg structures. The first alleles of both brm and osa were isolated because they fail to derepress the P2 promoter in the Antp Ns mutant. As noted by Kennison and Tamkun (1988) the trxG genes identified in their screen, including the osa gene, might regulate HOM gene function at a variety of different levels. They might regulate transcription or translation of the HOM genes, or encode cofactors that interact with the HOM proteins in regulating target genes. Since brm has been shown to affect HOM gene transcription, the genetic interaction with brm suggests that osa may also act at the level of HOM gene transcription. Antp proteins are normally not expressed in the cells that form the adult antenna. Misexpression of Antp proteins during the larval stage in these cells causes them to differentiate leg structures instead of antennal structures. The Antp Ns allele derepresses the Antp P2 promoter in the eye-antennal disc, expressing wild-type Antp transcripts from the Antp promoter. The penetrance of the antenna-to-leg transformation of Antp Ns mutants is greatly reduced in osa heterozygotes. High levels of osa expression are required only for the Antp P2 promoter, and not for the function of Antp proteins expressed from other promoters (Vázquez, 1999).

osa is also required maternally for proper embryonic segmentation. Although osa function appears to be important for expression of some HOM and segmentation genes in imaginal tissues, homozygous osa mutants die late in embryogenesis with no clear defects in either segmentation or segment identity. To determine whether wild-type maternal osa gene products deposited in the egg might be sufficient for segmentation and segment identity, homozygous germ cells were generated for the osa alleles that are strong Antp Ns suppressors. Loss of maternal osa functions has dramatic effects on the segmentation of the embryo. When rescued by a wild-type allele inherited from the father, the embryos secrete cuticle but have severe defects in segmentation, resembling mutants for the early-acting gap segmentation genes. When both maternal and zygotic osa functions are lacking, the embryos fail to differentiate any cuticle at all. The failure to detect obvious changes in the homozygous osa mutants from heterozygous mothers is clearly a consequence of the maternally encoded osa gene products, which function early in embryogenesis to activate transcription of target genes. Because of the severe defects in embryos lacking maternal osa function and the cascade of regulatory interactions between the segmentation and HOM genes early in embryogenesis, no attempt was made to identify the earliest-acting genes affected by loss of osa function (Vázquez, 1999).

Two regions of Osa have homology to other genes: within region I (residues 854 to 1104) there is a 97 amino-acid sequence (residues 993 to 1087) that contains a putative ARID (AT-rich interaction domain) that is conserved in the Drosophila Dead ringer (Dri) and mouse Bright proteins and in at least 10 other proteins. Although the Dri protein was identified in a screen for proteins that bound a consensus sequence for the EN homeodomain (Kalionis, 1993), Dri lacks any homology to the homeodomain (Gregory, 1996). The BRIGHT (B cell regulator of IgH transcription) protein binds to the minor groove of a consensus MAR (matrix attachment region) sequence. MARs organize chromatin fibers into looped domains by attachment to the nuclear matrix and may function as boundary elements for transcriptional domains. They may also collaborate with enhancers to generate extended domains of accessible chromatin. Dri and Bright are sequence-specific DNA binding proteins and the ARID domain is essential, but not sufficient for this binding. The consensus target sequences for Dri, Bright and En binding are very similar. Dri binds the PuATTAA sequence (Gregory, 1996); Bright binds the PuATa/tAA sequence, and En binds GATCAATTAAAT. All of these contain the same ATTAA core sequence (Vázquez, 1999).

Of the other 10 proteins reported to have an ARID domain, particular interest is found in the SWI1 protein, given the fact that it is a member of the SWI/SNF complex. The possibility that Osa might be the putative Drosophila SWI1 homolog was investigated. SWI1 has long tracks of polyasparagine, polyglutamine, and a putative Cys4 zinc-finger motif. Osa is very rich in proline but no zinc-finger motif is detected. SWI1 has in common with Osa clusters of sequence made up principally of only two or three amino acids. Very recently, a protein called p270 has been described as a member of the human BRG1 complex and has been proposed as a human SWI1 homolog (Dallas, 1998). p270, like OSA and SWI1, has glutamine-rich regions, an ARID domain and several copies of the LXXLL motif (where L is leucine and X is any amino acid). This motif mediates binding to nuclear receptors. Interestingly, Osa has three copies of this motif. Although Drosophila ESTs corresponding to proteins related to several yeast SWI/SNF subunits (including SWI2/SNF2, SWI3, SNF5, and SWP73) have been recovered, it is interesting to note that no EST corresponding to SWI1 has yet been identified. It is possible that OSA, SWI1 and p270 ARID-domain-containing proteins play similar roles in their respective organisms (Vázquez, 1999).

Is OSA essential for the function of the BRM complex? If so, one might expect brm and osa mutants to have identical phenotypes, and the mutation with the strongest effects in one assay should be the mutation with the stronger effects in all other assays. This is not observed. For example, there are much greater effects on Scr, Ubx, and Abd-B in brm heterozygotes than in osa heterozygotes, but the reverse is observed for Antp. Another important difference is the germ line requirements for brm and osa, i. e., brm clones do not make eggs while osa clones make normal appearing eggs that are fertilized but fail during embryogenesis. Thus, brm is required under conditions that do not appear to require osa. If Osa is a subunit of the BRM complex, it is not essential for all of the complex’s functions. Consistent with this possibility, the Osa protein was not identified as one of the major subunits of the BRM complex in the Drosophila embryo. However, it remains possible that Osa is a substoichiometric subunit of the BRM complex, or that it is associated with Brm at other stages of development. Another possibility is that the Osa protein targets the BRM complex to specific promoters (e.g., Antp P2). To date, no protein from the SWI/SNF complex (including SWI3 or the ARID-domain protein SWI1), has been shown to bind DNA in a sequence-specific manner (Vázquez, 1999 and references).

It is proposed that the Osa protein may be involved in the targeting of the BRM complex in Drosophila. Whether an intrinsic member of the BRM complex or merely an associated partner, the OSA protein may interact with specific target sequences in cis-regulatory elements to anchor or recruit the BRM complex. Given the patterns of expression driven by Antp cis-regulatory sequences in a reporter gene transposon, it is likely that there are En DNA-binding sites in the 10 kb region 5' to the Antp P2 promoter. Since the ARID domain found in the Osa protein may bind to En target sites, it is possible that Osa proteins will bind directly to these sequences. It is also possible that Osa may bind AT-rich regions of DNA with little specificity. The delineation of brm and osa response elements should allow a clarification of whether they act in concert or independently. It is also possible that the BRM complex alters chromatin structure in order to facilitate the binding of Osa to its target sites. Subsequent to this, Osa would act independently of the BRM complex to activate transcription (Vázquez, 1999 and references).

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).

The Drosophila BRM complex facilitates global transcription by RNA polymerase II

Drosophila brahma encodes the ATPase subunit of a 2 MDa complex that is related to yeast SWI/SNF and other chromatin-remodeling complexes. Brm was identified as a transcriptional activator of Hox genes required for the specification of body segment identities. To clarify the role of the Brm complex in the transcription of other genes, its distribution was examined on larval salivary gland polytene chromosomes. The Brm complex is associated with nearly all transcriptionally active chromatin in a pattern that is generally non-overlapping with that of Polycomb, a repressor of Hox gene transcription. Reduction of Brm function dramatically reduces the association of RNA polymerase II with salivary gland chromosomes. A few genes, such as induced heat shock loci, are not associated with the Brm complex; transcription of these genes is not compromised by loss of Brm function. The distribution of the Brm complex thus correlates with a dependence on Brm for gene activity. These data suggest that the chromatin remodeling activity of the Brm complex plays a general role in facilitating transcription by RNA polymerase II (Armstrong, 2002).

The Brm complex is localized to nearly every active gene in salivary gland nuclei, but is it required for transcription of these genes? brm is an essential gene; individuals homozygous for brm null alleles die before completing embryogenesis. To investigate whether brm is required for transcription in salivary gland nuclei, use was made of a GAL4-inducible transgene encoding a dominant-negative form of the Brm protein (BrmK804R). A single amino acid substitution in the highly conserved ATP-binding site of the Brm protein (lysine to arginine at residue 804) eliminates Brm ATPase activity. However, this mutant form of the Brm protein is properly assembled into the Brm complex and therefore antagonizes wild-type brm function in vivo. An hsp70-GAL4 driver under non-heat shock conditions was used to express BrmK804R in salivary glands. For simplicity, these individuals will be referred to as UASbrmK804R. Although the mutant salivary glands are reduced in size, this level of expression of BrmK804R does not drastically disrupt the structure of the chromosomes. The DAPI-stained chromosomes are slightly thinner than control chromosomes derived from control glands expressing LacZ, but otherwise display an overall normal banding pattern. To address whether Brm is necessary for transcription, the distribution of Pol IIoSer2 was examined on the mutant chromosomes. As one of the predominant forms of elongating Pol II in flies, the presence of Pol IIoSer2 on polytene chromosomes reflects active transcription. Upon BrmK804R expression in the salivary glands, the level of Pol IIoSer2 on chromosomes is drastically reduced. The Brm complex is not only required for the elongation of Pol II; the levels of initiating and promoter-paused Pol II (Pol IIa) were also reduced on mutant chromosomes. Thus, a functional Brm complex appears to be required for Pol II association with promoters (Armstrong, 2002).

Since brm is essential, it is possible that the observed reduction in Pol II transcription is a secondary consequence of a general decline in the level or activity of Pol II due to loss of an essential gene. Two experiments were conducted to exclude this possibility. First, the levels of Pol II present in the salivary glands was examined. The ratios of Pol II protein relative to a control protein, a-tubulin, are not reduced in larval salivary glands expressing BrmK804R when compared with glands expressing LacZ. Thus, partial loss of Brm function does not result in a specific decline in the levels of Pol II (Armstrong, 2002).

Secondly, to demonstrate that chromosomes derived from salivary glands expressing BrmK804R are still capable of a transcriptional response, the expression of a gene not regulated by Brm was examined. The heat shock genes were chosen for these experiments, because genetic studies have suggested that the Brm complex might not be required for transcription from the heat shock promoter. Furthermore, the Brm protein does not localize to the heat shock puffs following heat shock. Lastly, expression of a dominant-negative form of human BRG1 has no effect on heat shock-induced activation of hsp70. Thus, the heat shock genes appear to be good candidates as controls to determine whether or not salivary glands expressing BrmK804R are competent for transcription. The heat shock response is found to be intact in glands expressing BrmK804R, since heat shock results in the recruitment of similar levels of Pol IIoSer2 to heat shock loci in polytene chromosomes expressing either LacZ or BrmK804R. These results suggest that loss of Brm function does not result in a non-specific loss of Pol II activity. It is therefore concluded that the Brm complex is required for transcription of the majority of Pol II genes (Armstrong, 2002).

How does the Brm complex activate transcription? The results suggest that the Brm complex is required for a relatively early step in transcription, since partial loss of Brm function results in reduced levels of RNA Pol IIa on salivary gland polytene chromosomes. Brm may be required for the binding of transcriptional activators, assembly of the pre-initiation or promoter-paused complex, and/or recruitment of Pol II. Furthermore, the similar distributions of Brm and elongating Pol II (Pol IIoSer2) on salivary gland polytene chromosomes suggest that Brm might also facilitate transcriptional elongation. It is noteworthy that the hsp70 heat shock genes do not require the Brm complex for their expression. The hsp70 genes are unusual in that when uninduced the genes exist in a relatively nucleosome-free configuration with a paused RNA Pol II that has produced a short RNA transcript. This configuration may not depend upon the Brm complex for transcriptional activity; rather, the open architecture of these promoters may be a consequence of known interactions with the NURF chromatin-remodeling complex and factors residing upstream of heat shock genes (Armstrong, 2002).

The data suggest that the Brm complex recognizes some unique feature of active genes. Whether Brm physically associates with Pol II, as has been reported for yeast SWI/SNF, was investigated. Although the chromosomal distributions of Brm and Pol II proteins are similar, their levels vary dramatically from site to site, suggesting that Brm and Pol II are not present in the same protein complex. In agreement with this, Pol II was not detected in the purified Brm complex. Deletion of the Brm bromodomain does not alter the distribution of the Brm protein. It is concluded that the Brm protein does not preferentially associate with acetylated chromatin via its bromodomain. Given the importance of post-translational modifications of histone tails in gene expression, it will be interesting to explore other possible connections between histone modifying enzymes and the Brm complex (Armstrong, 2002).

The results are also consistent with the proposal that some chromatin-remodeling complexes act as global regulators of chromatin fluidity. In the nucleus, the mass of the Brm complex is equivalent to the mass of the histones. Perhaps the essential, abundant Brm complex acts globally to remodel nucleosomes and facilitate transcription. The regulation of this promiscuous complex may hinge upon negative acting factors that function to exclude the Brm complex from inappropriate genes. PC and the PcG proteins are good candidates for these factors. A core PRC1 protein complex (consisting of PC, PSC, PH and dRING1) prevents the human homolog of Brm (BRG1) from binding to chromatin in vitro. Since BRG1, PC and PSC are all capable of binding DNA, it has been proposed that this PcG complex might compete with the Brm complex for binding to the linker regions of chromatin. Alternatively, PRC1 might create higher order chromatin structures that are not accessible to the Brm complex. The predominantly non-overlapping distributions of PC and Brm on salivary gland polytene chromosomes are consistent with both of these models. However, these proposed mechanisms are more difficult to reconcile with the heat shock genes, which do not associate with Brm, yet are also not bound by PcG proteins (Armstrong, 2002).

The Drosophila trithorax group gene tonalli (tna) interacts genetically with the Brahma remodeling complex and encodes an SP-RING finger protein

The trithorax group genes are required for positive regulation of homeotic gene function. The trithorax group gene brahma encodes a SWI2/SNF2 family ATPase that is a catalytic subunit of the Brm chromatin-remodeling complex. The Drosophila tonalli (tna) gene was identified by genetic interactions with brahma. tna mutations suppress Polycomb phenotypes and tna is required for the proper expressions of the Antennapedia, Ultrabithorax and Sex combs reduced homeotic genes. The tna gene encodes at least two proteins, a large isoform (TnaA) and a short isoform (TnaB). The TnaA protein has an SP-RING Zn finger, conserved in proteins from organisms ranging from yeast to human and thought to be involved in the sumoylation of protein substrates. Besides the SP-RING finger, the TnaA protein also has extended homology with other eukaryotic proteins, including human proteins. tna mutations also interact with mutations in additional subunits of the Brm complex, with mutations in subunits of the Mediator complex, and with mutations of the SWI2/SNF2 family ATPase gene kismet. It is proposed that Tna is involved in postranslational modification of transcription complexes (Gutiérrez, 2003).

Flies heterozygous for some combinations of mutations in trithorax group genes have a held-out wings phenotype that results from reduced expression of the Antp P2 promoter. On the basis of this phenotype several dominant enhancers of brm were isolated. Two of the new mutations are alleles of the trithorax group gene taranis. These mutations, tara2 and tara20, show genetic interactions with multiple alleles of brm. In addition, one mutation was isolated in a second gene, tonalli (tna). tonalli means 'fate' in Náhuatl, an indigenous Mexican language. tna1 was mapped to polytene chromosome bands 67F3-4. By analyzing the available collection of P-element insertion lines from the BDGP three P-element insertion strains [P{PZ}l(3)rI075rI075, P{lacW}l(3)s0583/02, and P{lacW}l(3)rI075L6731] were identified that failed to complement tna1. These P-insertion mutations are referred to as tna2, tna3 and tna4, respectively (Gutiérrez, 2003).

The Antp gene has two alternative promoters, P1 and P2. The AntpNs allele derepresses the Antp P2 promoter in the eye-antennal disc and expresses wild-type Antp transcripts from the Antp promoter. Derepression of the Scr gene causes the appearance of extra sex combs on the second and third legs of males. This derepression can be caused by gain-of-function alleles of Scr, such as ScrMsc, or by loss-of-function mutations in Polycomb group genes, such as Pc3 or Pc4. Several trithorax group genes (including brm, mor, osa, kis, skd and kto) were first identified as suppressors of the extra sex combs phenotype caused by derepression of Scr or as suppressors of the antenna to leg transformation caused by derepression of Antp in the Nasobemia (Ns) allele of Antp. Since the tna gene was identified on the basis of genetic interactions with brm, tests were performed to see whether tna mutations could also suppress these two homeotic derepression phenotypes. It was found that all tna mutations strongly suppress the extra sex combs phenotype caused by Pc3, Pc4 or ScrMsc, but only weakly suppress the antenna to leg transformation caused by the AntpNs mutation (Gutiérrez, 2003).

Tests were performed to see if tna mutations can genetically interact with mutations in the trithorax group genes encoding subunits of the Brm or Kis chromatin remodeling complexes or the Mediator coactivator complex to give the same held-out wings phenotype observed in the brm/+; osal/+ transheterozygous combinations. Genetic interactions were sought between tna and several other trithorax group mutations that probably do not encode subunits of the Brm, Kis or Mediator complexes. tna1 shows strong genetic interactions with some mutations in the Brm complex (brm2, osa1, mor1 and mor2), with kis mutations (kis1 and kis13416), and with some mutations in the Mediator complex (skd2, skdlL7062 and skdrk760). There were no strong interactions with the snr10319 mutation in the Brm complex or the kto1 and Trap80s2956 mutations in the Mediator complex. No strong genetic interactions were observed with ash21, trx1, trx00347, urd2 or sls1 trithorax group mutations (Gutiérrez, 2003).

Northern blot analyses were prepared with RNA samples purified from different developmental stages using the ZAP1 cDNA clone as a probe. This clone was isolated from a lambdaZAP embryonic library and overlaps all of the tna translated exons. Two signals (6.1 and 4.2 kb) were found that correspond to major tna transcripts. The 6.1 kb transcript is present at all stages, but its expression increases at the second larval instar and reaches its maximum in the pupal stage. The 4.2 kb transcript was first detected in third instar larvae, but it is most abundant in the pupal and adult stages (Gutiérrez, 2003).

The Northern and sequence analyses of tna predict at least two alternative transcripts encoding products of 1109 and 610 residues. The long form of the protein (TnaA) is translated from 10 coding exons and may have three different amino termini. The mRNA for the short form (TnaB) lacks exons 5-8 and part of exon 9. Both proteins have similar amino termini, which have two Gln-rich regions, but they do not share the same carboxyl termini; the alternative splicing of the short form generates a frameshift that changes the open reading frame after the alternative splice. This frameshift generates a stop codon in the middle of exon 9. Exon 7 is present only in TnaA and encodes a possible bipartite nuclear location signal and an SP-RING (Siz/PIAS-RING) putative zinc finger (Gutiérrez, 2003).

Blast analyses of the TnA protein sequence identified four regions. Region I and IV (residues 1-494, and residues 799-1109, respectively) do not show homology to any other reported protein in any organism. Region I contains two blocks of glutamine residues. Region III (647-798) includes the SP-RING finger (residues 718-760), which is present in several proteins from organisms ranging from yeast to human. One family of SP-RING finger proteins are the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] family. One of the PIAS proteins, Miz1 (ARIP3/PIASXalpha) has also been identified as a cofactor of homeotic gene function in mice. In the Drosophila genome, the only other SP-RING finger proteins are ZimpA and ZimpB (zinc finger-containing, Miz1, PIAS3-like). The Zimp proteins belong to the PIAS family and are encoded by the Su(var)2-10 locus. Region III also includes the putative bipartite nuclear location signal (residues 668-686). The 300 amino acid domain spanning both Regions II and III identifies a new signature named the XSPRING (eXtended SP-RING finger) domain. The TnaB form shares regions I and II with TnaA, but has a unique carboxyl terminus. It does not show any additional homology to other known or predicted proteins (Gutiérrez, 2003).

It is concluded that the XSPRING domain may identify new group of human, mouse and Arabidopsis proteins and may be the signature for a new subgroup of SUMO E3 ligases within the PIAS family. SUMO (small ubiquitin-related modifier) is a ubiquitin-like protein (UBL) that is covalently attached to other proteins in a manner analogous to that of ubiquitin. Conjugation of SUMO-1 to all protein targets requires the E1-activating heterodimer Aos1/Uba2 (see Aos1) and the single E2-conjugating Ubc9 enzyme. The target specificity is conferred by the SUMO E3 ligases. There are at least two types of SUMO E3 ligases that are structurally unrelated. The first type is represented by the PIAS family of SP-RING finger proteins. The second type is represented by RanBP2, a nuclear pore complex protein. TnaA has an SP-RING finger within the larger XSPRING domain (Gutiérrez, 2003).

Although the role of sumoylation is not clear, it has been suggested that sumoylation could be an address tag for protein targeting. Most of the identified substrates of sumoylation are nuclear proteins, and the sumoylated forms are often found in specific subnuclear protein complexes. Preferential accumulation sites for sumoylated proteins are the PML nuclear bodies. PML, a protein found in PML nuclear bodies, is a RING-finger protein. Another core component of PML nuclear bodies is Sp100, a protein that interacts with HP1 and HMG1/2 families and a major cellular substrate for sumoylation. In vitro, sumoylated Sp100 has a higher affinity for the HP1 protein. Relocalization of proteins to nuclear bodies after sumoylation can modulate transcriptional activity. It has been suggested that nuclear bodies might stimulate SUMO conjugation, and that proteins transiently associated with nuclear bodies include SUMO targets. Thus, sumoylation can modulate the interaction of transcription factors with transcriptional corregulators (Gutiérrez, 2003).

The SUMO ligation target consensus sequence is PsiKxE (where Psi is an aliphatic residue) surrounding the substrate lysine(s) that is sumoylated. Although this consensus sequence is short, all of the proteins encoded by the trithorax groups genes that interact genetically with tna (including TnaA itself) have one or more blocks of this consensus sequence. However, some trithorax group genes that do not interact with tna, such as trithorax (trx), also encode proteins with the 'sumoylation consensus'. Sumoylation of the HDAC4 deacetylase is catalysed by the RanBP2 SUMO E3 ligase. While HDAC4 has several 'sumoylation consensus' sequences, only one functions in vitro and in vivo. The possibility that subunits of the Brm and/or Kismet complexes might be targets for sumoylation opens the window for a new level of regulation of the activity of chromatin remodeling complexes. This level of regulation could involve the modification of their subnuclear localization within the nucleus, although mutation of the SUMO acceptor site in HDAC4 did not change its subcellular distribution. Alternatively, sumoylation could target the homeotic function itself or its cofactors (Gutiérrez, 2003).

Another possible role for sumoylation is as an antagonist of ubiquitylation. Ubiquitylation is a key regulator of transcription and it has been suggested that sumoylation could be an inhibitor of ubiquitylation. The RING and PHD fingers have been described in proteins that have E3 ubiquitin ligase activities. In that sense it is intriguing that Trip-Br1 (the tara homolog in mice; (Hsu, 2001) was identified because it binds the PHD-bromodomain of Krip1/TIF1ß which also has an RBCC (RING finger-B boxes-coiled coil) RING finger. Krip1/TIF1ß has a dual role because it has been described as a corepressor of a subset of Krüppel-type zinc finger proteins and as a hormone-dependent coactivator that interacts with several nuclear hormone receptors. Mutations in a ubiquitin-conjugating enzyme (UbcD1) have been shown to affect homeotic gene silencing. Since tna mutations affect homeotic gene activation, antagonism between the ubiquitylation and sumoylation post-translational modifications may play a key role in homeotic gene regulation. Antagonism of ubiquitylation and targeting nuclear sublocalization are not mutually exclusive roles for sumoylation, and it is possible that both will be found to have roles in regulating the functions of chromatin remodeling and/or transcriptional co-activator complexes (Gutiérrez, 2003).

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).

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. The expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing were examined by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate the described methods for expression profiling as a way to analyze developmental programs, and they show that modifications to chromatin structure are key to changes in cell fate. These findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells (Klebes, 2005).

When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells in a proximodorsal region known as the 'weak point' can switch fate and transdetermine. These 'weak point' cells give rise to cuticular wing structures. The leg-to-wing switch is regulated, in part, by the expression of the vestigial (vg) gene, which encodes a transcriptional activator that is a key regulator of wing development. vg is not expressed during normal leg development, but it is expressed during normal wing development and in 'weak point' cells that transdetermine from leg to wing. Activation of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).

Sustained proliferation appears to be a prerequisite for fate change, and conditions that stimulate growth increase the frequency and enlarge the area of transdetermined tissue. Transdetermination was discovered when fragments of discs were allowed to grow for an extensive period of in vivo culture. More recently, ways to express Wg ectopically have been used to stimulate cell division and cell cycle changes in 'weak point' cells (Sustar, 2005), and have been shown to induce transdetermination very efficiently. Experiments were performed to characterize the genes involved in or responsible for transdetermination that is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because it is well characterized, it can be efficiently induced and it can be monitored by the expression of a real-time GFP reporter. These attributes make it possible to isolate transdetermining cells as a group distinct from dorsal leg cells, which regenerate, and ventral leg cells in the same disc, which do not regenerate; and, in this work, to directly define their expression profiles. This analysis identified unique expression properties for each of these cell populations. It also identified a number of genes whose change in expression levels may be significant to understanding transdetermination and the factors that influence developmental plasticity. One is lamina ancestor (lama), whose expression correlates with undifferentiated cells and is shown to control the area of transdetermination. Another has sequence similarity to the mammalian augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which controls regenerative capacity in the liver and is upregulated in mammalian stem cells. Fifteen regulators of chromatin structure [e.g. members of the Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated in transdetermining cells, and mutants in seven of these genes have significant effects on transdetermination. These studies identify two types of functions that transdetermination requires -- functions that promote an undifferentiated cell state and functions that re-set chromatin structure (Klebes, 2005).

The importance of chromatin structure to the transcriptional state of determined cells makes it reasonable to assume that re-programming cells to different fates entails reorganization of the Polycomb group (PcG) and trithorax group (trxG) protein complexes that bind to regulatory elements. Although altering the distribution of proteins that mediate chromatin states for transcriptional repression and activation need not involve changes in the levels of expression of the PcG and trxG proteins, the array hybridization data was examined to determine if they do. The PcG Suppressor of zeste 2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD to DWg/VWg comparisons, but the cut-off settings did not detect significant enrichment or repression of most of the other PcG or trxG protein genes with either clustering analysis or the method of ranking median ratios. Since criteria for assigning biological significance to levels of change are purely subjective, the transdetermination expression data was re-analyzed to identify genes whose median ratio changes within a 95% confidence level. Fourteen percent of the genes satisfied these conditions. Among these genes, 15/32 PcG and trxG genes (47%) had such statistically significant changes. Identification of these 15 genes with differential expression suggests that transdetermination may be correlated with large-scale remodeling of chromatin structure (Klebes, 2005).

To test if the small but statistically significant changes in the expression of PcG and trxG genes are indicative of a functional role in determination, discs from wild-type, Polycomb (Pc), Enhancer of Polycomb [E(Pc)], Sex comb on midleg (Scm), Enhancer of zeste [E(z)], Su(z)2, brahma (brm) and osa (osa) larvae were examined. The level of Wg induction was adjested to reduce the frequency of transdetermination and both frequency of transdetermination and area of transdetermined cells was determined. The frequency of leg discs expressing vg increased significantly in E(z), Pc, E(Pc), brm and osa mutants, and the frequency of leg to wing transdetermination in adult cuticle increased in Scm, E(z), Pc, E(Pc) and osa mutants. Remarkably, Su(z)2 heterozygous discs had no vg expression, suggesting that the loss of Su(z)2 function limits vg expression (Klebes, 2005).

Members of the PcG and trxG are known to act as heteromeric complexes by binding to cellular memory modules (CMMs). The functional tests demonstrate that mutant alleles for members of both groups have the same functional consequence (they increase transdetermination frequency). The findings are consistent with recent observations that the traditional view of PcG members as repressors and trxG factors as activators might be an oversimplification, and that a more complex interplay of a varying composition of PcG and trxG proteins takes place at individual CMMs. Furthermore the opposing effects of Pc and Su(z)2 functions are consistent with the proposal that Su(z)2 is one of a subset of PcG genes that is required to activate as well as to suppress gene expression. In addition to measuring the frequency of transdetermination, the relative area of vg expression was examined in the various PcG and trxG heterozyogous mutant discs. The relative area decreased in E(Pc), brm and osa mutant discs, despite the increased frequency of transdetermination in these mutants. There is no evidence to explain these contrasting effects, but the roles in transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that the transcriptional state of determined cells is implemented through the controls imposed by the regulators of chromatin structure (Klebes, 2005).

The determined states that direct cells to particular fates or lineages can be remarkably stable and can persist after many cell divisions in alien environments, but they are not immune to change. In Drosophila, three experimental systems have provided opportunities to investigate the mechanisms that lead to switches of determined states. These are: (1) the classic homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of cells to maintain homeotic gene expression, and (3) transdetermination. During normal development, the homeotic genes are expressed in spatially restricted regions, and cells that lose (or gain) homeotic gene function presumably change the transcriptional profiles characteristic of the particular body part. In the work reported here, techniques of micro-dissection, RNA amplification and array hybridization were used to monitor the transcription profiles of cells in normal leg and wing imaginal discs, in leg disc cells that regenerate and in cells that transdetermine from leg to wing. The results validate the idea that changing determined states involves global changes in gene expression. They also identify genes whose function may be unrelated to the specific fates of the cells characterized, but instead may correlate with developmental plasticity (Klebes, 2005).

Overlap between the transcriptional profiles in the wing and transdetermination lists (15 genes) and with genes in subcluster IV (high expression in wing discs) is extensive. The overlap is sufficient to indicate that the TD leg disc cells have changed to a wing-like program of development, but interestingly, not all wing-specific genes are activated in the TD cells. The reasons could be related to the incomplete inventory of wing structures produced (only ventral wing) or to the altered state of the TD cells. During normal development, vg expression is activated in the embryo and continues through the 3rd instar. Although the regulatory sequences responsible for activation in the embryo have not been identified, in 2nd instar wing discs, vg expression is dependent upon the vgBE enhancer, and in 3rd instar wing discs expression is dependent upon the vgQE enhancer. Expression of vg in TD cells depends on activation by the vgBE enhancer, indicating that cells that respond to Wg-induction do not revert to an embryonic state. Recent studies of the cell cycle characteristics of TD cells support this conclusion (Sustar, 2005), but the role of the vgBE enhancer in TD cells and the incomplete inventory of 'wing-specific genes' in their expression profile probably indicates as well the stage at which the TD cells were analyzed: they were not equivalent to the cells of late 3rd instar wing discs (Klebes, 2005).

Investigations into the molecular basis of transdetermination have led to a model in which inputs from the Wg, Dpp and Hh signaling pathways alter the chromatin state of key selector genes to activate the transdetermination pathway. The analyses were limited to a period 2-3 days after the cells switched fate, because several cell doublings were necessary to produce sufficient numbers of marked TD cells. As a consequence, these studies did not analyze the initial stages. Despite this technical limitation, this study identified several genes that are interesting novel markers of transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as several genes that function in the transdetermination process (e.g., lama and the PcG genes). The results from transcriptional profiling add significant detail to a general model proposed for transdetermination (Klebes, 2005).

(1) It is reported that ectopic wg expression results in statistically significant changes in the expression of 15 PcG and trxG genes. Moreover, although the magnitudes of these changes were very small for most of these genes, functional assays with seven of these genes revealed remarkably large effects on the metrics used to monitor transdetermination -- the fraction of discs with TD cells, the proportion of disc epithelium that TD cells represent, and the fraction of adult legs with wing cuticle. These effects strongly implicate PcG and trxG genes in the process of transdetermination and suggest that the changes in determined states manifested by transdetermination are either driven by or are enabled by changes in chromatin structure. This conclusion is consistent with the demonstrated roles of PcG and trxG genes in the self-renewing capacity of mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states. The results now show that the PcG and trxG functions are also crucial to pluripotency in imaginal disc cells, namely that pluripotency by 'weak point' cells is dependent upon precisely regulated levels of PcG and trxG proteins, and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).

The data do not suggest how the PcG and trxG genes affect transdetermination, but several possible mechanisms deserve consideration. A recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase of the cell cycle. Several proteins involved in cell cycle regulation physically associate with PcG and trxG proteins, and Brahma, one of the proteins that affects the metrics of transdetermination, has been shown to dissociate from chromatin in late S-phase and to reassociate in G1. It is possible that changes in the S-phase of TD cells are a consequence of changes in PcG/trxG protein composition (Klebes, 2005).

Another generic explanation is that transdetermination is dependent or sensitive to expression of specific targets of PcG and trxG genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in the Drosophila genome, one is in direct proximity to the vg gene. It is possible that upregulation of vg in TD cells is mediated through this element. Another factor may be the contribution of targets of Wg signaling, since targets of Wg signaling have been shown to be upregulated in osa and brm mutants. These are among a number of likely possible targets, and identifying the sites at which the PcG and trxG proteins function will be necessary if an understand is to be gained of how transdetermination is regulated. Importantly, understanding the roles of such targets and establishing whether these roles are direct will be essential to rationalize how expression levels of individual PcG and trxG genes correlate with the effects of PcG and trxG mutants on transdetermination (Klebes, 2005).

(2) The requirement for lama suggests that proliferation of TD cells involves functions that suppress differentiation. lama expression has been correlated with neural and glial progenitors prior to, but not after, differentiation, and it is observed that lama is expressed in imaginal progenitor cells and in early but not late 3rd instar discs. lama expression is re-activated in leg cells that transdetermine. The upregulation of unpaired in TD cells may be relevant in this context, since the JAK/STAT pathway functions to suppress differentiation and to promote self-renewal of stem cells in the Drosophila testis. It is suggested that it has a similar role in TD cells (Klebes, 2005).

(3) A role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that were found to be present in the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and 'community effects' regulate activation of the Notch pathway in TD cells (Klebes, 2005).

(4) The upregulation in TD cells of many genes involved in growth and division, and the identification of DNA replication element (DRE) sites in the regulatory region of many of these genes supports the observation that TD cells become re-programmed after passing through a novel proliferative state (Sustar, 2005), and suggests that this change is in part implemented through DRE-dependent regulation (Klebes, 2005).

There was an interesting correlation between transdetermination induced by Wg mis-expression and the role of Wg/Wnt signaling for stem cells. Wg/Wnt signaling functions as a mitogen and maintains both somatic and germline stem cells in the Drosophila ovary, and mammalian hematopoetic stem cells. Although the 'weak point' cells in the Drosophila leg disc might lack the self-renewing capacity that characterizes stem cells, they respond to Wg mis-expression by manifesting a latent potential for growth and transdetermination. It seems likely that many of the genes are conserved that are involved in regulating stem cells and that lead to disease states when relevant regulatory networks lose their effectiveness (Klebes, 2005).

The prevalence of transcription factors among the genes whose relative expression levels differed most in the tissue comparisons was intriguing. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In these studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but it is noted that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons nor universal (Klebes, 2005).

Genetic screens for enhancers of brahma reveal functional interactions between the BRM chromatin-remodeling complex and the delta-notch signal transduction pathway in Drosophila

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a 2-MDa chromatin-remodeling complex. brm was identified in a screen for transcriptional activators of homeotic genes and subsequently shown to play a global role in transcription by RNA polymerase II. To gain insight into the targeting, function, and regulation of the BRM complex, a screen was carried out for mutations that genetically interact with a dominant-negative allele of brm (brmK804R). First, dominant mutations were screened that are lethal in combination with a brmK804R transgene under control of the brm promoter. In a distinct but related screen, dominant mutations were identified that modify eye defects resulting from expression of brmK804R in the eye-antennal imaginal disc. Mutations in three classes of genes were identified in the screens: genes encoding subunits of the BRM complex (brm, moira, and osa), other proteins directly involved in transcription (zerknullt and RpII140), and signaling molecules (Delta and vein). Expression of brmK804R in the adult sense organ precursor lineage causes phenotypes similar to those resulting from impaired Delta-Notch signaling. These results suggest that signaling pathways may regulate the transcription of target genes by regulating the activity of the BRM complex (Armstrong, 2005).

A total of 17,146 mutant chromosomes were screened and 39 mutations were recovered that genetically interact with a dominant-negative allele of brm (brmK804R). Of the 25 mutations that were positively identified, nearly half (48%) are alleles of genes encoding subunits of the BRM complex (brm, mor, or osa), suggesting that the other genes identified in the screens are also critical for brm function. Similar screens could be used to study any Drosophila chromatin-remodeling factor that functions as the ATPase subunit of a protein complex (Armstrong, 2005).

The screens identified a single allele of RpII140, which encodes the second largest subunit of RNA pol II. Other alleles of RpII140 also dominantly enhanced eye defects resulting from expression of brmK804R. This finding complements the observation that the BRM complex is required for global transcription by RNA pol II and suggests that the BRM complex may interact more closely than previously thought with the general transcriptional machinery. These findings are consistent with the observation that yeast TFIID and RNA pol II are required for the recruitment of SWI/SNF to the RNR3 promoter. No physical interaction between RNA pol II and the BRM complex was detected by co-immunoprecipitation, however, and SWI/SNF recruitment does not depend upon RNA pol II at all yeast promoters. Why the basal transcription machinery targets chromatin-remodeling complexes to some, but not all, promoters remains to be determined (Armstrong, 2005).

Two distinct BRM complexes (called BAP and PBAP) have been identified in Drosophila (Mohrmann, 2005). Both complexes contain the BRM ATPase (related to the yeast SWI2/SNF2 and RSC ATPases), the SANT-domain protein Moira (MOR), the HMG-domain protein BAP111, the actin-related protein BAP55, actin, BAP60, and SNR1. The BAP complex contains Osa, while the PBAP complex lacks Osa and instead contains Polybromo (Baf180, CG11375) and the ARID-domain, zinc-finger protein BAP170. BAP may represent the Drosophila counterpart of the yeast SWI/SNF and human BAF complexes, while PBAP appears more highly related to the yeast RSC and human PBAF complexes (Mohrmann, 2005). Both BAP and PBAP are abundant and are widely associated with transcriptionally active chromatin in larval salivary glands. Both complexes use the BRM ATPase; the expression of BRMK804R should therefore interfere with the functions of both the BAP and PBAP complexes (Armstrong, 2005).

The presence or absence of the Osa subunit distinguishes the BAP complex from PBAP. Two osa alleles were isolated from the male-specific lethality screens, suggesting that this screen has the potential to identify factors important for BAP function. The osa alleles fail to modify the eye defects caused by expression of dominant-negative brm (as does a deficiency spanning osa), suggesting that the eye-based screen may select for genes important for PBAP function. In agreement with these observations, it has been found that while osa interacts with brm in the wing, it acts in opposition to brm in the eye. The elucidation of the relative roles of BAP and PBAP in vivo will require the isolation of mutations in genes encoding unique subunits of this complex, including polybromo and BAP170 (Armstrong, 2005).

Numerous recent studies have revealed close functional relationships between chromatin-remodeling complexes and histone-modifying enzymes. For example, the MOF histone acetyltransferase functionally antagonizes the Drosophila ISWI chromatin-remodeling factor; bromodomains within the yeast RSC chromatin-remodeling complex recognize acetylated histone H3 and methylation of lysines 4 and 9 of H3 and lysine 20 of H4 by Ash1 may recruit the BRM complex. Histone modification, including methylation of lysine 4 of H3, is also required for expression of Notch target genes (Armstrong, 2005).

However, to date no E(brm) mutations have been identified in genes encoding histone-modifying enzymes. Also no genes were recovered encoding structural components of chromatin or subunits of other chromatin-remodeling complexes. Why weren't mutations in these classes of genes recovered in these screens? Recover of mutations in histone genes was not expected in these screens since they are present in many copies in flies. The eye-based screen was limited to the third chromosomes, and genes on the X chromosome would have escaped detection in both of screens. Furthermore, it is not believed that either one of the genetic screens was taken to saturation. It is also possible that chromatin-remodeling and modifying enzymes that interact with brm are redundant or are not expressed in limiting quantities (Armstrong, 2005).

Dl represented the largest E(brm) complementation group; over a third of the mutations (36%) were alleles of Dl. These findings suggest that the functions of the BRM complex and the Notch signaling pathway are intimately related. Notch signaling is one of the most extensively studied signaling pathways. It is essential for the development of most tissues and is likely present in all metazoans, although this study focuses on the pathway in Drosophila. A transmembrane ligand (either Delta or Serrate) on the signaling cell binds the Notch receptor on the signal-receiving cell, resulting in two proteolytic cleavages of the Notch transmembrane protein. This proteolysis causes the release of the Notch ICD, which translocates to the nucleus to regulate gene expression. Once in the nucleus, the ICD forms a complex with the Suppressor of Hairless [Su(H)] transcription factor (a CSL protein) to activate Notch target genes. In the absence of signaling (and therefore the absence of ICD), Su(H) complexes with corepressors that deacetylate histones to repress transcription of target genes. The role of Notch signaling is particularly well understood in regard to cell fate determinations within the adult SOP lineage. Loss of Dl-Notch signaling can result in an increase of neurons or glia at the expense of other cell types (Armstrong, 2005).

Previous work suggested that the BRM complex is critical for the development of the peripheral nervous system; somatic clones of brm mutant tissue throughout the fly showed duplicated, stunted, or fused mechanosensory bristles. Expression of the dominant-negative allele of brm results in similar bristle defects, as well as alterations in the number and identities of campaniform sensilla, sensory organs used for flight. The identification of numerous alleles of Dl in these screens as well as the observation of increased penetrance of a variety of phenotypes in individuals heterozygous for alleles of both brm and Dl is consistent with these observations and points to a close functional connection between the Notch signaling pathway and the BRM complex (Armstrong, 2005).

To explore further the connection between the BRM complex and Dl-Notch signaling, the role of the BRM complex was investigated in cell fate specification within the adult SOP lineage, where every stage of development is regulated by Dl-Notch signaling. Reduced Dl-Notch signaling within the imaginal disc proneural cluster that gives rise to the SOP leads to formation of ectopic SOPs that form perfectly normal sense organs, leading to bristle/socket duplications, a phenotype similar to the bristle defects seen in brm mutant clones. In contrast, reduced Dl-Notch specifically within the SOP lineage results in loss of external cell types and production of ectopic internal cell types such as glia or neurons. This is precisely the phenotype observed following expression of brmK804R within the SOP lineage (Armstrong, 2005).

What is the role of the BRM complex in the Notch signaling pathway? Since the BRM complex plays a global role in transcription by RNA pol II, it is possible that the genetic interactions and phenotypes that were observed are the result of decreased Dl expression. This is thought unlikely due to the selectivity of the screens. Indeed, no genetic interactions were observed between Dl and RpII140 mutations. It is also possible that the BRM complex and the Dl-Notch pathway are independently regulating the same target genes. If both pathways are limiting, a reduction in Dl-Notch signaling may enhance a brm phenotype. A more intriguing possibility is that Dl-Notch signaling may regulate the activity or targeting of the BRM complex. As a ubiquitous complex that is critical for the transcription of most genes by RNA pol II genes, the BRM complex is a logical target for the signaling pathways. Once the ICD of Notch is in the nucleus, it may form complexes not only with Su(H), but also with the BRM complex, thus regulating its activity or its association with Notch target genes. Strong support for this model is provided by recent biochemical studies of the human BRM (hBRM) protein. hBRM physically interacts with the ICD of Notch and both hBRM and ICD are found to be associated with the promoters of Notch target genes (Kadam, 2003). On the basis of these findings, further analyses of the interactions between Dl-Notch signaling and the BRM chromatin-remodeling complex are clearly warranted (Armstrong, 2005).

The data suggest that the BRM complex may play an important role in another signal transduction pathway. An allele of vn, which encodes a secreted protein related to the mammalian neuregulin family of ligands for the EGF receptor, was recovered as an enhancer of eye defects resulting from the expression of brmK804R. Many signal pathways intersect and complex interactions between EGF receptor signaling and the Notch pathway have been reported in Drosophila. EGF receptor signaling can work in concert with or antagonistically to Notch signaling. The current findings suggest that the BRM complex interacts with one or both of these pathways during eye development, but the precise nature of these interactions remains to be determined (Armstrong, 2005).

In conclusion, unbiased genetic screens have led to an unexpected connection between the BRM chromatin-remodeling complex and Dl-Notch signaling. Both the BRM complex and the Dl-Notch signaling pathway are conserved in mammals; these results therefore suggest that similar interactions may be critical for mammalian development. In mice, loss of Notch activity leads to tumor formation; similarly the genes encoding subunits of the mammalian BRM complexes also act as tumor suppressors. Further work is required to determine the precise nature and extent of interactions between the BRM chromatin-remodeling complex and signaling pathways (Armstrong, 2005).

γTub23C interacts genetically with brahma chromatin-remodeling complexes in Drosophila melanogaster

The brahma gene encodes the catalytic subunit of the Drosophila BRM chromatin-remodeling complexes. Screening for mutations that interact with brahma, the dominant-negative Pearl-2 allele of γTub23C was isolated. γTub23C encodes one of the two γ-tubulin isoforms in Drosophila and is essential for zygotic viability and normal adult patterning. γ-Tubulin is a subunit of microtubule organizer complexes. This study shows that mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of microtubule organizer complexes, suppress the recessive lethality and the imaginal phenotypes caused by γTub23C mutations. The genetic interactions between γTub23C and chromatin-remodeling mutations suggest that γ-tubulin might have a role in regulating gene expression (Vázquez, 2008).

Proteins identified as part of the eukaryotic cytoskeleton may have more direct roles in transcriptional regulation than originally thought. Actin and actin-related proteins (ARPs) are found in BRM complexes from yeast to humans, including the BRM complexes in Drosophila. The function of actin and ARPs in these complexes is not well understood. Some ARPs interact with DNA-bending proteins and with histones and it was proposed that they facilitate chromatin architecture and interactions between complexes or function as histone chaperones. Actin is also part of preinitiation complexes and is necessary for transcription by RNA polymerases I, II, and III. The α- and/or β-tubulins are also found with a subset of trithorax-group proteins in the mammalian ASCOM complex (Activating signal cointegrator 2, Asc2 complex), which is required for transactivation by nuclear receptors, and in a histone H2A deubiquitinase complex. γ-Tubulin is essential for microtubule function, but unlike α- and β-tubulin, it is not a component of microtubules. Rather, it is located at microtubule-organizing centers (MTOCs) and functions in the initiation of microtubule nucleation and in the establishment of microtubule polarity. γ-Tubulin contributes to the proper formation of mitotic spindles and cytoplasmic microtubular arrays. There are critical cytoskeletal and nuclear envelope connections, linking, for example, MTOCs to the nuclear lamina. In addtion, γ-tubulin has been proposed to have microtubule- and/or centrosome-independent function(s) in mitosis or spindle assembly checkpoints (Vázquez, 2008).

Drosophila embryonic γ-tubulin exists in two related complexes: a large complex similar to the Xenopus γTuRC (γ-tubulin ring complex) (36.9S, ~2000 kDa) and a small soluble complex called γTuSC (γ-tubulin small complex) (8.5S ~240 kDa). The Drosophila γTuRC consists of approximately eight polypeptides, including γ-tubulin, Grip163, Grip128, Grip91, Grip84, Grip75, and GP71WD. The γTuRC has a lockwasher-like structure and a cap at one of the ends of the complex. The Drosophila γTuSC is a tetramer of two γ-tubulin molecules and one molecule each of Grip91 and Grip84. Several γTuSCs form the γTuRC lockwasher region. The other Grips (Grip163, 128, and 75) form the cap (Vázquez, 2008).

Drosophila is the only metazoan in which the genes encoding subunits of the γTuSC and γTuRC complexes have been functionally studied using genetic approaches. Null mutations in dd4 (which encodes Grip91) and in Grip84 are lethal and display defects in spindle assembly (Barbosa, 2003; Colombié, 2006), while null mutations in Grip128 and Grip75 are viable, but sterile (Vázquez, 2008).

In Drosophila there are two γ-tubulin genes, γTub23C and γTub37C. They encode very similar (but not identical) proteins, but they have different expression patterns and mutant phenotypes. γTub37C is largely restricted to the female germline and early stages of embryogenesis. It is required for bicoid (bcd) mRNA localization at mid-oogenesis, female meiosis, and nuclear proliferation. In syncytial embryos, γTub23C is in the soluble small γTuSC fraction and is absent at the centrosome. At this stage, γTub37C is found in both γTuSC and γTuRC fractions. It is localized at the centrosome and over the spindle regions. γTub37C mutants are female sterile (Vázquez, 2008).

The γTub23C isoform is expressed in a variety of tissues in both sexes, including larval brains and imaginal discs, and it is required for somatic mitotic divisions. It is also expressed in ovaries and is the only isoform expressed in testes. γTub23C is required for meiosis in males and for spermatogenesis (Vázquez, 2008).

The γTub23CPl-2 mutation was isolated in a mutant screen designed to identify genes that interact with brm in wing development. In addition to showing genetic interactions with brm, γTub23CPl-2 mutants are homozygous lethal, while the heterozygotes have defects in imaginal eye and wing development. γTub23CPl-2 is a dominant-negative mutation and l(2)23Ce alleles are loss-of-function mutations in γTub23C with recessive phenotypes similar to the dominant phenotypes of γTub23CPl-2. γTub23C has 30% identity to α- and β-tubulins, which are structural components of microtubules. It is known which parts of the β-tubulin protein are involved in autoregulation for translation and for binding and hydrolysis of GTP. The γ-tubulin protein shares some of these regions with β-tubulin. The γTub23C mutations characterized in this work do not map to any of these known regions, with the exception of the truncated form in the γTub23CA15-2 allele. This suggests that the proteins synthesized from the γTub23CA14-9, γTub23CA6-2, γTub23CPl-2, and γTub23Cbmps1 alleles might affect other γ-tubulin functions (Vázquez, 2008).

It was a surprise to identify a dd4 allele with no discernible phenotype except the suppression of some γTub23C mutant phenotypes (including zygotic lethality). Since dd4 encodes Grip91, a protein that physically interacts with γ-tubulin, it is believed that the genetic interactions have important implications (Vázquez, 2008).

Grip91, Grip84, and γ-tubulin form the lockwasher region of γTuRC and γTuSC complexes. Grip91 and Grip84 (or their orthologs in yeast and humans) interact with each other and with γ-tubulin. The interactions between Grip91 and γ-tubulin facilitate binding of GTP to γ-tubulin. Grip91 is required for correct bipolar spindle assembly during mitosis and male meiosis and it helps to locate γ-tubulin to the centrosome (Vázquez, 2008).

Grip91 is an essential protein encoded by the dd4 gene. Semilethal alleles have held-up wings and other imaginal defects and are male sterile. The dd4su(Pl) allele is unusual in that it has no defects in viability, fertility, or developmental patterning. Its only phenotype is the suppression of class I (but not class II) genotypes of γTub23C (Vázquez, 2008).

What is the significance of the two types of γTub23C alleles from the functional point of view? The defects produced by suppressible alleles may involve γTuSC and/or γTuRC functions, while the defects produced by nonsuppressible alleles may involve γTub23C functions independent of the γTuSC and γTuRC complexes. It is also possible that different mutant proteins, although in some cases retaining partial activity, may affect other different functions of γTub23C. Some of these other functions may require Grip91 (and possibly the integrity of γTuRC and/or γTuSC complexes) and some may not. Such functions could affect the assembly of the γTuSC and/or γTuRC complexes, the transport of the complex(es) to subcellular compartments, and/or the relationships of γTub23C with other proteins involved in microtubule-independent processes. It is believed that the new alleles of γTub23C and dd4 that have been characterized can help to test the current structural models of γTuRC and γTuSC complexes proposed in biochemical and crystallographic studies (Vázquez, 2008).

Recent work shows that γ-tubulin has a microtubule-independent role in establishing or maintaining a mitotic checkpoint block (Prigozhina, 2004) and that γTuRCs proteins may have a centrosome-independent role in the spindle assembly checkpoint. For this latter function, γ-tubulin is probably in a complex associated with Cdc20 and BubR1 kinases (Muller, 2006). This study found that the genetic interactions between γTub23C and Brm are caused not by reduced γTub23C transcription, but more probably by the presence of defective γ-tubulin proteins. This suggests roles for γ-tubulin in transcription and/or chromatin remodeling. This is further supported by the recent description of interactions between Pericentrin (an integral centrosomal component) and CHD3, a Brm-related protein in the NuRD chromatin-remodeling complex (Vázquez, 2008).

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 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 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).

Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells

In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. This study reports that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation (He, 2014. PubMed ID: 25068272).

Drosophila Brahma complex remodels nucleosome organizations in multiple aspects

ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. Brm was knocked down in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions (Shi, 2014).

brahma: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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