brahma

Targets of Activity

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

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

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

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 is a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).

Protein Interactions

The SNR1 and BRM proteins are present in a large [> 2 x 10(6) Da] complex, and they co-immunoprecipitate from Drosophila extracts (Dingwall, 1995).

The SWI/SNF complex in yeast and Drosophila is thought to facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression. The mechanism by which it is targeted to specific genes is poorly understood and may involve direct DNA binding and/or interactions with specific or general transcription factors. A mammalian complex has been purified by using antibodies against BRG1, a human homolog of SWI2/SNF2. This complex is likely functionally related to the yeast SWI/SNF complex because all five subunits identified so far (referred to as BAFs, for BRG1-associated factors) are homologs of the yeast SWI/SNF subunits. Nevertheless the mammalian complex may be functionally more related to the abundant yeast complex known as Rsc because of its high abundance in the cell. The yeast Rsc complex, is distinct from the yeast SWI/SNF complex, although the yeast Rsc subunits identified so far are all homologous to the yeast SWI/SNF subunits. The 57-kDa subunit (BAF57), which is present only in higher eukaryotes but not in yeast, has now been cloned. BAF57 is shared by all mammalian complexes and contains a high-mobility-group (HMG) domain adjacent to a kinesin-like region. Both recombinant BAF57 and the whole complex bind four-way junction (4WJ) DNA, which is thought to mimic the topology of DNA as it enters or exits the nucleosome. Surprisingly, complexes with mutations in the HMG domain of BAF57 can still bind 4WJ DNA and mediate ATP-dependent nucleosome disruption. This work describes the first identified DNA binding subunit for SWI/SNF-like complexes and suggests that the mechanism by which mammalian and Drosophila SWI/SNF-like complexes interact with chromatin may involve recognition of higher-order chromatin structure by two or more DNA binding domains. A partial Drosophila cDNA shows significant homology to human BAF57 (70% identity and 90% similarity within the HMG domain). Preliminary data suggest that the cDNA encodes the homologous subunit of BAF57 within the Drosophila SWI/SNF-like complex (Wang, 1998).

Site-directed mutagenesis was used to investigate the functions of conserved regions of the Brm protein. Domain II is essential for brm function and is required for the assembly or stability of the Brm complex. This 62 amino acid domain of Brm (residues 549-610) is located N-terminal to the ATPase domain, and is 48% identical to the corresponding region of SWI2/SNF2. This domain is also conserved in the putative human homologs of Brm, BRG1, and hbrm (83% identity to Brm in both proteins). The two-hybrid system has revealed an interaction between this domain and the SWI3 subunit of the SWI/SNF complex. Although a Drosophila homolog of SWI3 has not yet been identified, proteins related to SWI3 are present in the human BRG1 and hbrm complexes. These observations strongly suggest that domain II of the Brm protein interacts with an as yet unidentified Drosophila relative of SWI3. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the Brm protein has no discernible phenotype (Elfring, 1998).

To determine if Brm physically interacts with other trithorax group proteins, the Brm complex was purified from Drosophila embryos and its subunit composition analyzed. The Brm complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the Brm complex are not encoded by trithorax group genes. The proteins that consistently copurify with Brm have been designated Brm-associated proteins (BAPs) and are referred to by their molecular mass in kDa (BAP45, BAP47, BAP55, BAP60, BAP74, BAP111 and BAP155). Two different purification schemes identify the same set of seven polypeptides associated with Brm. Western blotting identified BAP45 as Snr1. The BAP155 protein is highly related to the BRG1/hBRM associated factors (BAFs) BAF155 and BAF170, and the yeast SWI3 and RSC8 proteins. Common to all of these proteins are three domains of unknown function: regions I, II and III. The 440 residues between the N terminus of BAP155 and domain I are highly conserved in the human BAF155 and BAF170 proteins (39% and 34% identity, respectively), but not in the yeast SWI3 and RSC8 proteins. SWI3 and RSC8 also lack the proline-rich domains immediately C-terminal to domain III that are present in BAP155 and its human counterparts. The BAP60 protein is highly related throughout its length to BAF60a, BAF60b and BAF60c, the human homologs of the yeast SWP73 and RSC6 proteins. BAP60 is most closely related to BAF60a (72% identity), which is consistent with the characterization of BAF60c as a potentially tissue-specific subunit and with the identification of BAF60b as a component of a variant 500 kDa complex in mammals. BAP60 is equally related to both yeast SWP73 and RSC6 proteins (approximately 16%-28% identity); however, the yeast proteins contain two relatively large insertions within the approximately 370 amino acid segment conserved in their Drosophila and human relatives. Thus the BRM complex contains four subunits (BRM, BAP155, BAP60 and BAP45/SNR1) that are conserved in the human BRG1 and hBRM complexes and in both the yeast SWI/SNF and RSC complexes (Papoulas, 1998 and references).

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

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

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

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

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

Multiple genes related to BRM, BAP155, BAP60 and BAP45/SNR1 are present in yeast and humans. To determine whether similar heterogeneity might exist in Drosophila, a search was carried out for additional genomic sequences related to BAP genes by Southern blotting. After hybridization and washing under low-stringency conditions, all major bands detected were consistent with restriction maps of the BAP cDNAs; no crosshybridizing bands were detected for BAP155, BAP60, BAP55 or SNR1. By contrast, a brm genomic DNA fragment crosshybridizes under these conditions to ISWI, a divergent ATPase related to BRM. Several additional weak signals were observed with the BAP111 probe. The BAP111 probe spans the HMG domain and may be detecting other HMG-Box genes. None of these weak signals persist after washing at high stringency. The Drosophila genome thus does not appear to contain multiple genes that could give rise to the subunit heterogeneity reported for the yeast and human counterparts of the BRM complex (Papoulas, 1998).

Biochemical evidence is presented for the existence of two additional complexes containing trithorax group proteins: a 2 MDa Ash1 complex and a 500 kDa Ash2 complex. Based on their genetic properties, three of the best candidates for trx-G members that physically interact with Brm are Absent, small or homeotic discs 1 and 2 (Ash1 and Ash2), and Trithorax. In spite of being bona fide members of the trx-G, neither Ash1, Ash2 nor Trithorax are found to be a part of the Brm complex. Affinity-purified polyclonal antibodies against Ash1 detect three prominent bands in embryo extracts, the largest of which is 270 kDa. The predicted size of the Ash1 protein (244 kDa) and the variability in amount of the smaller bands detected in different experiments argues that the 270 kDa band represents full-length Ash1 and that the smaller bands are degradation products. Affinity-purified antibodies against ASH2 detect a single band of 94 kDa. Although the Brm, BAP45/Snr1, Ash1 and Ash2 proteins are readily detected by western blotting in whole embryo extracts, neither the Ash1 nor Ash2 proteins are detected in purified Brm complex. Similar experiments using antibodies against Trx did not yield reproducible results, presumably due to the low abundance and instability of this >350 kDa protein. An examination to see if Ash1 or Ash2 are physically associated with Brm in embryo extracts used a coimmunoprecipitation assay. Neither Ash1 nor Ash2 were found to coimmunoprecipitate with Brn. It is therefore concluded that the Ash1 and Ash2 proteins do not stably interact with the Brm complex. To determine whether Ash1 and Ash2 are components of protein complexes distinct from the Brm complex in the Drosophila embryo, the native molecular mass of both proteins was examined by gel filtration chromatography. The ASH1 protein has a native molecular mass of approximately 2 MDa. By contrast, Ash2 has an apparent native molecular mass of approximately 500 kDa. No monomeric Ash1 or Ash2 is detected in embryo extracts. It is concluded that the Drosophila embryo contains at least three distinct protein complexes containing trx-G proteins: the 2 MDa BRM complex, a 2 MDa Ash1 complex and a 500 kDa Ash2 complex (Papoulas, 1998).

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

The Drosophila osa gene, like yeast SWI1, encodes an AT-rich interaction (ARID) domain protein. Genetic and biochemical evidence is presented that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. To determine whether Osa is associated with the high molecular weight Brm complex, Schneider cell nuclear extracts were fractionated through a glycerol gradient and immunoblotted with antibodies against the various proteins. Osa, Brm and Snr1 co-sediment in the bottom third of the gradient, suggesting that they are part of a large protein complex. Although Osa and Brm are present in similar fractions, Snr1 sediments in the bottom half of the gradient and could also be part of another complex that does not contain Osa or Brm. Alternatively, the anti-Snr1 antibody might be much more sensitive, detecting very low levels of the Snr1 protein. When glycerol gradient fractions are immunoprecipitated with anti-Osa antibody, Osa, Brm and Snr1 co-precipitate in the same region of the gradient in which they co-sediment. ISWI and Ash2 both show broad sedimentation patterns, appearing in the bottom half of the gradient, but neither protein is immunoprecipitated from the gradient fractions with anti-Osa antibody. Thus, in vivo, Osa is found in a large complex with Brm and Snr1, but does not bind to proteins in other chromatin remodeling complexes. The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. Some Brahma-containing complexes do not contain Osa and Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex (Collins, 1999).

osa genetically interacts with trithorax group genes. Ectopic expression of a dominant-negative form of Brm with a mutation in the ATP binding site (UAS-brm^K804R) disrupts many developmental processes. An optomotor-blind (omb)-GAL4 driver was used to direct expression of UAS-brm^K804R in the central region of the wing disc; this results in loss of the distal wing margin, formation of ectopic campaniform sensillae and wing margin bristles, and disruptions in wing vein morphology. These phenotypes are strongly enhanced in animals heterozygous for osa. Expression of UAS-brm^K804R at the wing margin using vestigial (vg)-GAL4 results in the loss of the proximal, posterior wing margin, a phenotype that is again enhanced in osa heterozygotes. The effect of increasing osa dosage was tested by co-expressing a full-length osa transcript under the control of the same vg-GAL4 driver, and this completely rescues the dominant-negative Brm phenotype. Interestingly, ectopic expression of osa alone with vg-Gal4 induces a dominant loss of proximal wing hinge structures, and this phenotype is also rescued in animals co-expressing osa and dominant-negative brm. This suggests that the functions of Osa and Brm are closely related, because a reduction in the activity of one can compensate for an excess of the other (Collins, 1999).

Ectopic expression of Osa in eye imaginal discs using eyeless (ey)-GAL4 results in a variable reduction in eye size. Rather than the expected suppression, an enhancement of this phenotype has been observed in flies that either co-express dominant-negative Brm or are heterozygous for brm. The eye phenotype is also enhanced by mor and SNF5-related 1 (Snr1), both of which encode components of the Brm complex. However, reducing the dosage of the trithorax group genes trx, ash1 or ash2 does not enhance the Osa overexpression phenotype. As expected, a reduction in osa dosage suppresses the small eye phenotype. Clones of mor mutant cells in the eye disc exhibit a severe reduction in growth, which is partially rescued if the cells are also mutant for osa. Taken together, these data demonstrate that osa shows strong and specific genetic interactions with components of the Brm complex. However, in the wing, osa appears to act in concert with brm, whereas in the eye, osa opposes the functions of brm, snr1 and mor (Collins, 1999).

The Drosophila trithorax group gene brahma encodes the ATPase subunit of a SWI/SNF-like chromatin-remodeling complex. A key question about chromatin-remodeling complexes asks how they interact with DNA, particularly in the large genomes of higher eukaryotes. This study reports the characterization of BAP111, a Brm-associated protein that contains a high mobility group (HMG) domain predicted to bind distorted or bent DNA. The presence of an HMG domain in BAP111 suggests that it may modulate interactions between the Brm complex and chromatin. BAP111 is an abundant nuclear protein that is present in all cells throughout development. By using gel filtration chromatography and immunoprecipitation assays, it has been found that the majority of BAP111 protein in embryos is associated with the Brm complex. Furthermore, heterozygosity for BAP111 enhances the phenotypes resulting from a partial loss of brm function. These data demonstrate that the BAP111 subunit is important for BRM complex function in vivo (Papoulas, 2001).

Brm and associated proteins have been purified to near homogeneity from Drosophila embryos. BAP111 is one of seven prominent copurifying proteins designated BAPs (BRM-associated proteins). Northern blotting with DNA fragments derived from the BAP111 EST clone LD13023 identifies a 3-kb transcript in Drosophila embryos. Sequencing of this clone and overlapping EST clones generates a 2,649-nt cDNA sequence that matches the predicted transcript (CT21811) of the Drosophila gene CG7055 (Papoulas, 2001).

The BAP111 RNA contains a single long ORF with an in-frame termination codon 21 nucleotides upstream of the initiating methionine. This ORF encodes a 749-aa polypeptide. BLAST searches reveal a strong homology between amino acids 83-170 of BAP111 and the HMG domains of a large number of proteins. HMG domains form a three-helix DNA-binding domain that binds the minor groove with relatively low affinity but is capable of recognizing or inducing bends in DNA. HMG domains fall into two groups: those that bind DNA nonspecifically and those that bind to specific sequences. HMG domains of the sequence-specific class are usually found in transcriptional regulators, including LEF-1, SRY, and the Sox family of proteins. These proteins contain a single HMG domain as well as a transcriptional activation or repression domain. By contrast, the non-sequence-specific HMG domains are highly abundant nonhistone chromosomal proteins considered to have a more architectural role; they shape DNA to facilitate the function of other factors. Such structural roles frequently require multiple HMG domains within a single polypeptide (Papoulas, 2001).

BAP111 shows equal overall sequence similarity to both the sequence-specific and nonspecific classes of HMG domains. This degree of similarity is likely to reflect requirements for DNA binding and bending that are common to both sequence-specific and nonspecific HMG domains. Like the sequence-specific class, BAP111 contains a single HMG domain. However, the critical residues used by HMG domains to recognize specific DNA sequences are generally hydrophilic, but in BAP111, the residues are hydrophobic. Furthermore, the BAP111 HMG domain shares three extremely conserved residues (Pro-89, Lys-146, and Tyr-149) with HMG domains of the nonspecific class. Thus, based on its sequence, BAP111 is likely to bind distorted or bent DNA without sequence specificity (Papoulas, 2001).

Another member of the nonspecific class of HMG-domain proteins, SSRP1, has been implicated in chromatin-based regulation of transcription. SSRP1 is a subunit of the human histone chaperone complex FACT (facilitates chromatin transcription), which is required for efficient elongation on chromatin templates. Although this functional similarity is intriguing, BAP111 is no more related to SSRP1 than to other HMG-domain proteins. Furthermore, BAP111 does not show particular homology to any of the recognized subgroups of nonspecific HMG domains, indicating that it may define a new subgroup of nonspecific HMG domains (Papoulas, 2001).

A small number of HMG-domain proteins are strikingly related to BAP111 outside the HMG domain, suggesting that they may be functional homologs. These HMG-domain proteins include the human and mouse BAF57 proteins, the zebrafish protein identified by the EST fe48d03.y1, and the predicted C. elegans protein g-III-342. Comparison of these proteins reveals a segment (amino acids 198-270) with an even greater degree of conservation than the HMG domain. This segment has been designated the NHRLI domain based on a conserved block of amino acids within the heart of this domain. This domain is 74% identical between Drosophila BAP111 and human BAF57 over 73 aa. The last 19 residues of the NHRLI domain overlap a region previously predicted to form a coiled-coil structure in BAF57. Computer predictions of the BAP111 structure using COILS confirmed the presence of this putative coiled-coil region in the BAP111 protein. By using the PHDSEC program, the HMG domain and the coiled-coil region were predicted to be helical. There is no strong prediction of structure for the initial 54 aa of the NHRLI domain. The remainder of BAP111, including the proline-rich C terminus (30.5% proline over 390 aa), has no significant similarity to any known sequences. The evolutionary conservation of the HMG and NHRLI domains suggests that they are critical for the function of the BAP111 protein, with the rest of the molecule having either dispensable or species-specific functions. No potential homologs of BAP111 are present in S. cerevisiae, suggesting that this subfamily of HMG-domain proteins is unique to higher eukaryotes. Thus, it is possible that BAP111 is involved in an aspect of chromatin remodeling that is unique to metazoa (Papoulas, 2001).

There are ~100,000 copies of the Brm complex per cell, or roughly 1 molecule per 20 nucleosomes. Because BAP111 is a stoichiometric subunit of purified Brm complexes, it is at least equally abundant. To verify that BAP111 is a subunit of the Brm complex, as opposed to a copurifying contaminant, the association between BAP111 and Brm was examined by using a coimmunoprecipitation assay. Anti-HA antibodies immunoprecipitate both Brm and BAP111 from extracts prepared from Drosophila embryos expressing HA-tagged BRM protein. These data confirm that BAP111 is a bona fide subunit of the BRM complex (Papoulas, 2001).

Vertebrate hBRM/BRG1 complexes contain a number of tissue-specific subunits. The expression of BAP111 parallels that of Drosophila Brm throughout embryonic, larval, pupal, and adult life. BAP111, like Brm, is a nuclear protein. Closer examination of dividing cells in early embryos revealed that BAP111 and Brm diffuse throughout the cell as the nuclei break down for mitosis and are not associated with the condensed metaphase chromosomes. No association of BAP111 with larval salivary-gland polytene chromosomes could be detected. BAP111, like BRM, is expressed ubiquitously throughout embryogenesis and appears to be enriched during later stages of embryogenesis in rapidly dividing tissues such as the central nervous system, as is true for Brm. Thus, BAP111 does not appear to be a stage-or tissue-specific subunit of the Brm complex (Papoulas, 2001).

Reduction of BAP111 function can modify the phenotypes that result from a partial loss of brm function. The replacement of a conserved lysine by an arginine in the ATP-binding site of the Brm protein eliminates the activity of the Brm protein without disrupting its assembly into the Brm complex. This brm^K804R mutation behaves, therefore, as a strong dominant-negative allele. Expression of brm^K804R in a variety of tissues antagonizes the function of endogenous Brm protein. Expression of a GAL4-responsive brm^K804R transgene (UAS-brm^K804R) under control of the eyeless driver (ey-GAL4) results in adults with slightly smaller or rough eyes. Furthermore, a small percentage of ey-GAL4 UAS-brm^K804R individuals die late in pupal development, presumably because of leaky expression of GAL4 in non-eye tissues (Papoulas, 2001).

If the strong genetic interaction between the BAP111 deficiency and brm^K804R is caused by the loss of BAP111, as opposed to one of the other genes in the deficiency, the expression of wild-type BAP111 should block the enhancement of brm^K804R-dependent phenotypes. A GAL4-responsive transgene (P[w⁺, UAS-BAP111] 22-1) was generated to express the full-length BAP111 protein. This transgene blocks the ability of the BAP111 deficiency to enhance brm^K804R phenotypes. These data confirm that loss of BAP111, and not some other gene within Df(1)18.1.15, is responsible for the enhancement of brm^K804R (Papoulas, 2001).

To investigate whether the HMG domain is essential for BAP111 function in vivo, a strain was generated bearing a GAL4-responsive transgene encoding a mutant protein that lacked 68 aa of the HMG domain. An Actin5C-GAL4 driver was used to ubiquitously express epitope-tagged wild-type or BAP111DeltaHMG protein in Drosophila embryos. The function of the HMG domain was examined by using a genetic assay. Unlike wild-type BAP111, BAP111DeltaHMG does not fully rescue the eye defects caused by the expression of brm^K804R in BAP111 hemizygotes. The HMG domain is important, therefore, for BAP111 function in vivo. However, BAP111DeltaHMG is able to rescue the pupal lethality caused by expression of brm^K804R in BAP111 hemizygotes, indicating that deletion of the HMG domain does not completely eliminate BAP111 function. Therefore, other conserved domains of BAP111, including the NHRLI domain, warrant further investigation (Papoulas, 2001).

How might BAP111 contribute to the function of the Brm complex? Because the HMG domain of BAP111 is likely to bind DNA, it is possible that it mediates interactions between the Brm complex and a subset of its target genes, a function similar to that proposed for the ARID protein OSA. For example, BAP111 might recognize an unusual chromatin structure present at particular target loci. Alternatively, a promoter-specific transcription factor might recruit the Brm complex, but efficient chromatin remodeling might require stabilization of the recruitment by the nonspecific DNA-binding affinity of BAP111. A third possibility is that BAP111 is involved not in gene-specific recruitment of the Brm complex, but rather in the catalytic event itself. For example, BAP111 might bind to transiently distorted DNA to stabilize a chromatin-remodeling intermediate. Further studies will be necessary to uncover the roles of this somewhat unconventional HMG-domain protein (Papoulas, 2001).

Wnt-induced formation of nuclear Tcf-ß-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of ß-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. An interaction between ß-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodeling complexes, is demonstrated. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, genetic interactions are observed between the Brg-1 and ß-catenin homologues in flies. It is concluded that ß-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodeling as a prerequisite for transcriptional activation (Barker, 2001).

Development in flies was selected as a model system to gain evidence for a functional interaction between Brg-1 and ß-catenin in vivo, assuming that this interaction would be conserved between mammals and Drosophila. It was asked whether reducing the gene dosage of brahma, the founder of the Brg-1 gene family, affects the mutant phenotypes caused by activation or depletion of Armadillo. First, a strain (GMR.Arm*) was used in which a constitutively activated form of Armadillo is overexpressed in the larval eye disc. The mutation in Arm* mimics the oncogenic point mutation S45F in the putative GSK3ß phosphorylation site of ß-catenin that renders the latter constitutively active. Oncogenic forms of ß-catenin such as this are potent transcriptional coactivators of Tcf. Flies bearing GMR.Arm* show rough and slightly glazed eyes whose size is reduced compared with the wild-type, due to late onset of apoptosis in the pupal disc caused by Arm* and dTcf. This phenotype is independent of armadillo gene dosage, but is reversed considerably towards wild-type in dTcf heterozygotes, whose gene dosage is reduced by half. This rough eye phenotype was reversed even further towards wild-type in brahma heterozygotes. Finally, a similar phenotypic suppression was observed in flies heterozygous for moira, a gene encoding another component of the Brahma complex. It is concluded that the mutant eye phenotype caused by activated Armadillo is as sensitive to the levels of Brahma complex components as it is to dTcf levels, indicating that the Brahma complex is required for the activity of Arm* (Barker, 2001).

It was also asked whether heterozygosity of Brahma complex genes would affect the mutant wing phenotype caused by Armadillo depletion in the wing disc. In the wing, armadillo is required for the integrity of the margin, and sequestration of Armadillo at the membrane by overexpression of the intracellular domain of cadherin (Arm^under) in the posterior wing disc causes extensive notches in the posterior wing. This phenotype is worsened by heterozygosity for activating genes of the Wingless pathway, and suppressed by heterozygosity of antagonists of this pathway. In particular, in Arm^under flies heterozygous for armadillo the posterior wing margin is completely absent, and the posterior wing area is much reduced. Likewise, dTcf heterozygotes showed on average slightly narrower wings, and less residual posterior margin than Arm^under controls. This modifying effect of dTcf is much milder than that observed in the eye, perhaps reflecting a dual function of dTcf in the wing margin (activating as well as repressing) similar to that observed in the embryonic cuticle. Significantly, brahma heterozygotes show considerably narrower wings than the controls. Indeed, brahma heterozygosity enhances the wing margin phenotype as strongly as armadillo heterozygosity. Finally, a slight worsening of this phenotype is also observed in moira heterozygotes. These genetic experiments in flies indicate functional interactions between Brahma complex genes and Armadillo/dTcf. Consistent with this, it has been reported that embryos derived from near-sterile brm transheterozygous mothers show reduced expression of dTcf target genes such as Ultrabithorax and engrailed (Barker, 2001).

Taken together, these fly genetic data support the conclusions from experiments in mammalian cells that the Brg-1 complex contributes to the activity of the ß-catenin-Tcf transcription factor (Barker, 2001).

Groucho corepressor proteins, which repress Tcf target gene activity in the absence of Wnt signaling, are known to recruit histone deactylases and are likely to effect repression by altering chromatin structure. Additionally, a recent study has demonstrated a role for SWI/SNF-mediated chromatin remodeling of Tcf target gene promoters in ensuring effective repression of gene activity in the absence of ß-catenin during fly development. Potentially, Groucho proteins in complex with Tcf could recruit Brahma complexes to target gene promoters through an interaction mediated by the histone deacetylase rpd3. The data support a mechanism in which ß-catenin accumulation following Wnt signaling promotes the formation of ß-catenin-SWI/SNF (or -Rsc) complexes in the nucleus, in competition with Groucho repressor complexes. In cooperation with the histone-acetylating activity of ß-catenin-bound p300/CBP, Brg-1-associated complexes would then remodel the chromatin structure of target gene promoters into a conformation more accessible to the basal transcription machinery, enhancing transactivation of target genes and leading to cellular responses. The initially paradoxical observation that chromatin-remodeling complexes are required for both the activation and repression of perhaps the same set of target genes can be resolved by the finding that in vitro, SWI/SNF and Rsc can catalyse both forward and reverse nucleosome remodeling reactions (Barker, 2001).

Histone chaperone ASF1 cooperates with the Brahma chromatin-remodeling machinery

De novo chromatin assembly into regularly spaced nucleosomal arrays is essential for eukaryotic genome maintenance and inheritance. The Anti-Silencing Function 1 protein (ASF1) has been shown to be a histone chaperone, participating in DNA-replication-coupled nucleosome assembly. Mutations in the Drosophila asf1 gene derepress silencing at heterochromatin and the ASF1 protein has a cell cycle-specific nuclear and cytoplasmic localization. Using both genetic and biochemical methods, it has been demonstrated that ASF1 interacts with the Brahma (SWI/SNF) chromatin-remodelling complex. These findings suggest that ASF1 plays a crucial role in both chromatin assembly and SWI/SNF-mediated chromatin remodelling (Moshkin, 2002).

Assembly of newly synthesized DNA into chromatin requires both nucleosome assembly activities and ATP-dependent chromatin-remodelling. Nucleosome assembly is the process by which newly synthesized histones are loaded onto naked DNA. This function is performed primarily by histone chaperones like Chromatin Assembly Factor-1 (CAF-1) and Nucleosome Assembly Protein-1 (NAP-1). However, nucleosome assembly factors alone are unable to efficiently produce long and regularly spaced nucleosomal arrays. To perform this function properly requires the recruitment of ATP-dependent chromatin-remodelling factors (Moshkin, 2002).

The asf1 gene was originally identified in yeast by its ability, when overexpressed, to repress silencing at the HMR and HML mating-type loci and at telomeres. Interestingly, it has also been shown that loss-of-function mutations in the yeast asf1 gene derepress transcription from silenced loci, when combined with mutations in the largest subunit of the yeast CAF-1 complex. Because of this, the role of ASF1 in silencing is thought to be in the assembly of silenced chromatin (Moshkin, 2002).

Recently, ASF1 has been shown to participate in the process of nucleosome assembly during DNA replication. Both biochemical and genetic studies have shown that ASF1 acts as a histone chaperone (Tyler, 1999, 2001; Munakata, 2000), which in concert with another histone chaperone, CAF-1, is thought to deposit histones H3 and H4 tetramers onto naked DNA. The assembly of nucleosome particles is completed by the addition of two dimers of histones H2A and H2B, probably by the histone chaperone, NAP-1 (Moshkin, 2002).

Although most studies on ASF1 have focused on its role in nucleosome assembly, recent data have shown that the yeast ASF1 is required for the proper transcriptional repression and activation of the histone genes (Sutton, 2001). This role in transcription raises the possibility that ASF1 may play a role in chromatin remodelling, as well as nucleosome assembly. This study explores the function of ASF1 in chromatin dynamics; ASF1 is shown to directly associated with the Brahma chromatin-remodelling machinery in flies (Moshkin, 2002).

During an EMS saturation screen over the deficiency Df(3L)kto2, which removes the 76BD region of the third chromosome, two mutations were identified in Drosophila asf1 gene (asf1¹ and asf1²). The asf1¹ mutation deletes two nucleotides in the open reading frame (ORF) at base pair 380 relative to the 'start' codon, creating a premature 'stop' codon and resulting in the truncation of approximately half of the ASF1 protein. The protein synthesized from asf1¹ mutant allele seems to be unstable. Although this protein still contains major epitopes recognized by polyclonal anti-ASF1 antibodies, it cannot be detected in crude protein extracts from heterozygous asf1¹ embryos. Hemizygous asf1¹ mutants are embryonic or larval lethal; loss of maternal ASF1 function completely blocks oogenesis as revealed by asf1¹ germ-line clones (Moshkin, 2002).

The asf1² removes 24 nucleotides from the ORF of asf1 at base pair 54 after the 'start' codon, resulting in an 8-amino-acid deletion in the protein. Because of the slight size difference between the mutant and wild-type proteins, it was not possible to determine whether the ASF1² protein is present in heterozygous embryos. Histone-binding experiments, however, indicate that the mutated ASF1 protein produced by asf1² allele shows markedly reduced binding to Drosophila histones H3 and H4 (Moshkin, 2002).

Because ASF1 is involved in the assembly of silenced chromatin in yeast (Tyler, 1999; Sharp, 2001), tests were performed to see whether ASF1 is able to affect the silenced chromatin state at pericentric heterochromatin. The In(1)w^m4h and In(1)w^m4 mutant lines, which carry an inversion on the X chromosome juxtaposing the white gene to centromeric heterochromatin, were used. This inversion leads to a classic position effect variegation (PEV) phenotype. The cell-autonomous inactivation of the white gene is thought to occur via the occasional spreading of the heterochromatic compaction of the DNA into the white gene. In flies heterozygous for the asf1¹ or asf1² mutations, it was observed that the white gene expression is strongly derepressed in comparison to flies carrying two wild-type asf1 alleles. The dominant suppression of PEV caused by mutations in the asf1 gene strongly suggests a function for ASF1 in the formation of silenced chromatin in Drosophila (Moshkin, 2002).

To gain more insight into ASF1 cellular function an antibody directed against the full-length ASF1 protein was raised and affinity purified. This antibody recognizes a single band of 26 kD in embryonic nuclear and crude extracts, which coincides with the predicted size of ASF1 and the size of bacterially expressed ASF1 protein (Moshkin, 2002).

ASF1 localization on polytene chromosomes was examined. ASF1 is strongly associated with multiple sites along the polytene chromosomes. Among them are many decondensed and transcriptionally active regions such as interbands and developmental puffs. Besides this, there is distinct staining of the chromocenter and the partially heterochromatic fourth chromosome, supporting the role of ASF1 in heterochromatin-mediated gene silencing. A particularly strong signal was observed at the 39DE region. The 39DE region is the location of the histone gene cluster. Interestingly, ASF1 is known to be involved in the control of the histone genes expression in yeast, and the staining of the 39DE region may point to a similar role in flies (Moshkin, 2002).

The intracellular localization of ASF1 protein was examined in the early Drosophila embryo. During the first hours of development, embryos undergo 13 cycles of nearly synchronous accelerated mitotic nuclear divisions, in which the G1 and G2 phases of the cell cycle are eliminated and cells only go through the S and M phases. Immunostaining with the anti-ASF1 antibody of these early embryos reveals that during S phase, ASF1 protein is primarily concentrated in the nucleus with only diffuse cytoplasmic staining. Because staining of the interphase cells of the salivary gland shows that nuclear ASF1 is associated with the chromosomes, it is likely that the early S phase embryonic staining is also chromosomal. Upon the commencement of mitosis, however, ASF1 nuclear staining fades and is not detected on the condensed chromatin (Moshkin, 2002).

To further explore ASF1 function in the regulation of chromatin dynamics and to identify potential interacting partners, the eyeless-GAL4, UAS-Asf1 strain was created, which over-expresses asf1 cDNA in the eye. This strain has a rough-eye phenotype, which allows an assay of genetic interactions between asf1 and genes known to be involved in the regulation of chromatin structure such as the Polycomb Group (PcG) and the Trithorax Group (TrxG) genes. Among the tested mutations [brm¹, brm², mor¹, osa², Df(3R)red-P6, kto¹, tara^L4, Asx^Xf23, ph⁴¹⁰, Pc³, Pcl^D5, Psc¹, E(z)^Su301], it was found that only mutations in the brahma (brm), moira (mor), and osa (osa) genes suppress the ASF1-mediated rough-eye phenotype. Interestingly, the proteins encoded by these genes are parts of the Brahma chromatin-remodelling complex (Moshkin, 2002).

To confirm the genetic interaction between ASF1 and the Brahma complex, a reciprocal analysis was performed. Transgenic flies overexpressing a dominant-negative form of brm (brm^K804R) in the eye were used; this results in a rough-eye phenotype, similar to asf1 overexpression. In this assay, brm and mor mutations aggravate the effect of brm^K804R over-expression, substantiating the dominant-negative nature of the brm^K804R allele. Similarly, the asf1¹ mutation significantly enhances the rough-eye phenotype caused by overexpression of the dominant-negative brm^K804R allele. These two complementary genetic assays strongly suggest that ASF1 functions in vivo in the Brahma chromatin-remodelling pathway (Moshkin, 2002).

Because the genetic data show that ASF1 acts in the Brahma chromatin-remodelling pathway, whether ASF1 directly interacts with the Brahma complex was tested. Although the ASF1 protein is not found tightly associated with a highly purified Brahma complex, the BRM and its associated MOR proteins are coimmunoprecipitated with anti-ASF1 antibodies from embryonic nuclear extracts suggesting that ASF1 does physically interact with the Brahma chromatin-remodelling complex. To test whether ASF1 can bind directly to the Brahma complex, GST pull-down experiments were performed using a bacterially expressed and purified ASF1-GST fusion protein and purified Brahma complex. Western blot analysis of pulled down material reveals that BRM, the ATPase subunit of the Brahma complex, is among the ASF1-interacting molecules, suggesting that ASF1 binds directly to the Brahma complex (Moshkin, 2002).

Therefore, Drosophila ASF1 plays a role in the formation of silenced chromatin similar to its yeast counterpart (Tyler, 1999). Although it is not yet clear how this is accomplished, the data re-emphasize the importance of chromatin assembly factors in the formation of silenced chromatin. Because regularly spaced nucleosomal arrays are a landmark of silenced heterochromatin, it is believed that ASF1 contributes to silencing through its nucleosome assembly activity (Tyler, 1999). Therefore, the reduction of silencing in asf1¹ mutants may result from the disruption of the nucleosome array at heterochromatin. This interpretation is supported by the chromocentric localization of the ASF1 protein on polytene chromosomes (Moshkin, 2002).

ASF1 protein has a cell cycle-specific chromosomal and cytoplasmic localization reminiscent of another histone chaperone protein, NAP-1. It has been speculated that the NAP-1 localization pattern could reflect a role for NAP-1 in binding newly synthesized histones in the cytoplasm and delivering them to the sites of chromatin assembly and/or remodelling. It is believed that ASF1 may play a similar role in histone shuttling to sites of chromatin assembly (Moshkin, 2002).

Furthermore, the data suggest a dualistic function for the histone chaperone ASF1 in both histone deposition during chromatin assembly and histone displacement during chromatin-remodelling. ASF1 interacts genetically and biochemically with the Brahma chromatin-remodelling complex. The Drosophila Brahma complex is a member of the SWI/SNF ATP-utilizing chromatin-remodelling factors conserved in yeast, flies, and mammals. Since the Brahma complex participates in both the initiation and the repression of transcription, it is believed that ASF1 may also function in transcriptional control. Although a direct role for ASF1 in transcription has not been firmly established, recent evidence supports this hypothesis: (1) mutation of the yeast asf1 gene results in the suppression of S-phase-specific histone genes activation (Sutton, 2001); (2) it was shown that ASF1 interacts with bromodomain-containing subunits of TFIID (Moshkin, 2002 and references therein).

The association of ASF1 with the chromatin-remodelling machinery raises several intriguing possibilities for ASF1 function in chromatin-remodelling. As a histone chaperone, ASF1 could facilitate chromatin-remodelling by attenuating the strong electrostatic histone-DNA contacts, in effect, lubricating the chromatin for remodelling factors. Recently, it has been shown that the disruption of a single histone-DNA contact by a mutation in the SIN domain of histone H4 results in an increased rate of remodelling by the yeast SWI/SNF complex. In a similar fashion, ASF1 may weaken the contacts of histones H3 and H4 with DNA, creating an altered nucleosome structure favorable for translocation by remodelling factors (Moshkin, 2002 and references therein).

However, ASF1 could function in targeting chromatin-remodelling factors to the sites of newly assembled chromatin. Since assembly of long and regularly spaced nucleosome arrays cannot be achieved by histone chaperones alone and some chromatin assembly complexes contain ATP-dependent nucleosome spacing activity, an interaction between ASF1 and chromatin-remodelling factors could indicate a mechanism by which functional chromatin is assembled after DNA replication (Moshkin, 2002).

Drosophila cyclin E interacts with components of the Brahma complex

Cyclin E-Cdk2 is essential for S phase entry. To identify genes interacting with cyclin E, a genetic screen was carried out using a hypomorphic mutation of Drosophila cyclin E (DmcycE^JP), which gives rise to adults with a rough eye phenotype. Among the dominant suppressors of DmcycE^JP, brahma (brm) and moira (mor) were identified. These genes encode conserved core components of the Drosophila Brm complex that is highly related to the SWI-SNF ATP-dependent chromatin remodeling complex. Mutations in genes encoding other Brm complex components, including snr1 (BAP45), osa and deficiencies that remove BAP60 and BAP111 can also suppress the DmcycE^JP eye phenotype. Brm complex mutants suppress the DmcycE^JP phenotype by increasing S phases without affecting DmcycE protein levels. DmcycE physically interacts with Brm and Snr1 in vivo. These data suggest that the Brm complex inhibits S phase entry by acting downstream of DmcycE protein accumulation. The Brm complex also physically interacts weakly with Drosophila retinoblastoma (Rbf1), but no genetic interactions were detected, suggesting that the Brm complex and Rbf1 act largely independently to mediate G₁ arrest (Brumby, 2002).

Several recent studies have provided strong connections between metazoan SWI- SNF complexes and regulation of the cell cycle. In yeast, the SWI- SNF complex is not essential for viability, and whole genome analyses of swi/snf mutants have shown roles in activation and repression of transcription. A screen for modifiers of E2F1/DP function in Drosophila identified new alleles of brm and mor as enhancers of the rough eye phenotype associated with ectopic expression of E2F1 and DP in the developing Drosophila eye imaginal disc. In support of this, mammalian homologs of Brm and Mor (hBrm/Brg1 and BAF55, respectively) have been reported to be present in cyclin E complexes and to be phosphorylated by cyclin E- Cdk2. Significantly, human homologs of Brm (hBrm and Brg1) inhibit entry into S phase and achieve this at least in part by cooperation with the tumor suppressor Rb. Furthermore, Rb can bind to Brg1 and hBrm, and the ability of Rb to induce G1 arrest has been shown to depend upon hBrm and Brg1 (Brumby, 2002 and references therein).

The genetic interactions with DmcycE or E2F1/DP and Brm complex genes initially were thought to be due to effects on DmcycE transcription or E2F/DP-dependent transcription, given the role of the Brm complex in transcriptional regulation. Surprisingly, the results of this study suggest that the Brm complex functions downstream of DmcycE transcription and protein accumulation. (1) No significant effect on DmcycE protein levels in DmcycE^JP eye discs was observed when the dosage of brm or mor was halved. (2) The rough eye phenotype due to overexpression of DmcycE from the GMR driver is enhanced by halving the dosage of brm and mor, indicating that Brm and Mor act to inhibit S phase entry downstream of DmcycE transcription. (3) DmcycE forms a complex with Brm and Snr1. Taken together, these data provide strong evidence that the Brm complex does not inhibit the G₁ to S phase transition by acting to down-regulate DmcycE transcription (Brumby, 2002).

It is also likely that the Brm complex does not act to down-regulate E2F1/DP-dependent gene transcription, since no effect was observed for at least two E2F1/DP targets in brm mutants. Thus, mutations in Brm complex genes suppress the DmcycE^JP mutant phenotypes by allowing progression into S phase without increasing either DmcycE protein levels or the expression of E2F1/DP-dependent genes. This suggests that one function of the Drosophila Brm complex is to restrict entry into S phase by inhibiting DmcycE-Cdk2 activity or by acting downstream of DmcycE-Cdk2 function. A function for Brm downstream of DmcycE-Cdk2 is consistent with reports that mammalian cyclin E can bind to and phosphorylate components of the Brm complex and thereby inactivate it. Thus the Brm complex may be acting as a curb to S phase entry that needs to be overcome by phosphorylation and inactivation by cyclin E-Cdk2 (Brumby, 2002).

Consistent with studies in cultured mammalian cells, the Rbf1 protein was found to be present in complexes with Brm or Snr1 in larval and embryonic extracts. However, in embryos, only a small portion of total cellular Rbf1 is present in Snr1 immunoprecipitates, in contrast to a significant fraction of the cellular DmcycE, suggesting that most Brm complexes do not contain Rbf1. The observation that Drosophila Rbf1 and Brm form a complex in vivo is consistent with studies in mammalian cells showing that hBrm and/or Brg1 can bind to and cooperate with Rb in transcriptional repression, and that hBrm and Brg1 are required for Rb-induced G₁ arrest. However, in Drosophila, no clear evidence was obtained for cooperation of brm or mor with rbf1 in S phase entry. It is possible that the phenotypes being examining were not sensitive enough for S phase effects to be observed. However, the lack of a strong effect of Brm complex mutants on the rbf1 mutant S phase phenotype, when strong genetic interactions were observed with Brm complex genes and DmcycE, suggests that Rbf1 and Brm primarily function independently in negatively regulating S phase entry. Therefore, the suppression of the S phase defect of DmcycE^JP by Brm complex mutants may not involve rbf1. Independent roles for Brm and Rb are also likely in mammalian cells since Rb knockout mice have a different mutant phenotype from that of Brg1 or Brm knockouts (Brumby, 2002).

In mammalian cells, Rb can form a complex containing both Brg1 and Hdac1, which is required to repress DmcycE transcription and may also have a role at replication origins. However, reducing the dose of the Drosophila Hdac gene, rpd3, did not suppress the DmcycE^JP rough eye phenotype. It is possible that no interaction was observed for rpd3 and DmcycE, because there are a least three other Hdacs in flies that may perform overlapping functions with rpd3. However, mutations in sin3a, which encodes a Hdac-interacting protein, enhance the DmcycE^JP rough eye phenotype, suggesting that Sin3a functions in opposition to Brm in regulating DmcycE or S phase entry. Further studies using specific mutations in other Drosophila Hdacs, and Hdac-interacting proteins are required to analyze further their role in the G₁ to S phase transition (Brumby, 2002).

How does the Brm complex mediate negative regulation of the G₁ to S phase transition? The results suggest that the Brm complex is playing a role independent of DmcycE transcription and E2F/DP-dependent transcription in negatively regulating the G₁ to S phase transition. One way in which this may occur is by transcriptional regulation of other critical G₁/S phase genes. For example, there is evidence that in Drosophila, the Brm complex is important in negatively regulating Armadillo-dTCF target genes in the Wingless signaling pathway. Although as yet there have been no studies showing directly that G₁/S phase-inducing genes are targets of the Wingless signaling pathway in Drosophila, this is possible based on studies in mammalian cells. Furthermore, the Wingless pathway clearly has a role in cell proliferation in some Drosophila tissues. Whether this is the mechanism by which the Brm complex mediates negative regulation of cell cycle entry requires further investigation (Brumby, 2002).

Another way in which the Brm complex may function is by restricting or regulating access to chromosomal origins of replication. Several studies have shown that ATP-dependent chromatin remodeling is important for modulating the initiation of chromosomal DNA replication. The data are consistent with the view that the Brm complex may play a role in this process, possibly functioning to restrict entry into S phase by acting directly to remodel nucleosomes at replication origins. In this scenario, DmcycE-Cdk2 may then act to phosphorylate and inactivate the Brm complex, allowing assembly or function of the pre-replication complex and replication origin firing. Indeed, cyclin E-Cdk2 has been shown to be recruited by the Cdc6 pre-replication complex protein to replication origins at the G₁ to S phase transition (Brumby, 2002).

Intriguingly, recent studies have shown that the E2F/DP complex also acts directly at replication origins. In the amplification of the chorion gene clusters during the ovarian follicle cell endoreplicative cycles, it has been shown that E2F1/DP is important in localizing the origin of replication complex specifically to the chorion gene origins and activating replication, and that Rbf1 is important in limiting DNA replication. This mechanism is not limited to these specialized cycles, since transcription-independent roles for E2F1 in inducing S phase have also been documented in the eye imaginal disc. Taken together, these studies suggest that the E2F1/DP-Rbf1 complex plays a non-transcriptional role in S phase by acting directly at DNA replication origins. In mammalian cells, a similar non-transcriptional role for Rb in DNA replication inhibition has been demonstrated, possibly through its functional association with the pre-replication complex protein Mcm7 and its localization to replication foci (Brumby, 2002).

Given the data for a role for Rb-E2F/DP directly at replication origins and the evidence that chromatin remodeling is important in replication initiation, it is possible that Brm and Rbf1 may both have a role at replication origins to prevent premature origin firing in G₁. However, the failure to detect a genetic interaction between brm complex genes and rbf1 suggests that they also have other important roles, independent of each other, in the G₁ to S phase transition (Brumby, 2002).

In summary, these results have shown that mutations in genes encoding components of the Brm chromatin remodeling complex can dominantly suppress a DmcycE hypomorphic allele by increasing the number of S phase cells without affecting cyclin E protein levels. Consistent with this view, DmcycE physically interacts with Brm and Snr1. Although a complex was also observed between the Brm complex and Rbf1, no genetic interactions have been detected between Brm complex genes and rbf1, suggesting that Rbf1 and Brm function largely independently in negatively regulating the G₁ to S phase transition. Taken together, these data suggest that the Brm complex negatively regulates entry into S phase, possibly in partial collaboration with Rbf1, and that this negative regulation can be abrogated by the action of cyclin E at the G₁ to S phase transition (Brumby, 2002).

Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes

The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. This study reports the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (1) the BAP complex, defined by the presence of Osa and the absence of Polybromo and BAP170, and (2) the PBAP complex, containing Polybromo and BAP170 but lacking Osa. Determination of the genome-wide distributions of Osa and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. It is concluded that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. These results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes (Mohrmann, 2004).

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