Snf5-related 1

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 ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and Trithorax (Trx) is a 150-aa motif, highly conserved during evolution. Yeast two hybrid screening of a Drosophila cDNA library was performed and interaction was detected between a Trx polypeptide spanning SET and the Snr1 protein. Snr1 is a product of snr1, which is classified as a trx group gene. Parallel interaction is found in yeast between the SET domain of ALL-1 and the human homolog of Snr1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 is detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalize with approximately 50% of Trx binding sites. Because Snr1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the observed interactions suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions of the C-terminal domains of ALL-1 and Trx with INI1/Snr1 (Rozenblatt-Rosen, 1998).

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).

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).

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).

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).

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).

dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, full-length dDYRK2 (Drosophila DYRK2) was used as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co-immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102 in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling (Kinstrie, 2006. Full text of article).

DEVELOPMENTAL BIOLOGY

Embryonic

The spatial and temporal patterns of expression of snr1 are similar to those of brm. The highest level of mRNA occurs in unfertilized eggs and early embryos. The level decreases until the end of embryogenesis, when little SNR1 mRNA is detected. Early in development SNR1 mRNA is found ubiquitously but later expression is confined to the ventral cord (CNS) and brain (Dingwall, 1995).

Effects of Mutation or Deletion

The snr1 gene is essential for normal development; genetically it interacts with brm and trithorax, suggesting cooperation in regulating homeotic gene transcription. Both snr1 and brm mutations, suppresses mutations in Polycomb (Dingwall, 1995).

The Drosophila Brahma (brm) complex, a counterpart of the Saccharomyces cerevisiae SWI/SNF ATP-dependent chromatin remodeling complex, is important for proper development by maintaining specific gene expression patterns. The Snr1 subunit is strongly conserved with yeast SNF5 and mammalian INI1 and is required for full activity of the brm complex. A temperature-sensitive allele of snr1 has been identified, caused by a single amino acid substitution in the conserved repeat 2 region, implicated in a variety of protein-protein interactions. Genetic analyses of snr1^E1 reveal that it functions as an antimorph and that snr1 has critical roles in tissue patterning and growth control. Temperature shifts show that snr1 is continuously required, with essential functions in embryogenesis, pupal stages, and adults. Allele-specific genetic interactions between snr1^E1 and mutations in genes encoding other members of the Brm complex suggest that snr1^E1 mutant phenotypes result from reduced Brm complex function. Consistent with this view, Snr1^E1 is stably associated with other components of the Brm complex at the restrictive temperature. Snr1 can establish direct contacts through the conserved repeat 2 region with the SET domain of the homeotic regulator Trithorax, and Snr1^E1 is partially defective for functional Trx association. Since truncating mutations of INI1 are strongly correlated with aggressive cancers, these results support the view that Snr1, and specifically the repeat 2 region, has a critical role in mediating cell growth control functions of the metazoan SWI/SNF complexes (Merenda, 2003).

SNR1 is an essential subunit of the Drosophila Brahma (Brm) ATP-dependent chromatin remodeling complex, with counterparts in yeast (SNF5) and mammals (INI1). Increased cell growth and wing patterning defects are associated with a conditional snr1 mutant, while loss of INI1 function is directly linked with aggressive cancers, suggesting important roles in development and growth control. The Brm complex is known to function during G₁ phase, where it appears to assist in restricting entry into S phase. In Drosophila, the activity of DmcycE/CDK2 is rate limiting for entry into S phase and the Brm complex can suppress a reduced growth phenotype associated with a hypomorphic DmcycE mutant. The results reveal that SNR1 helps mediate associations between the Brm complex and DmcycE/CDK2 both in vitro and in vivo. Further, disrupting snr1 function suppresses DmcycE^JP phenotypes, and increased cell growth defects associated with the conditional snr1^E1 mutant are suppressed by reducing DmcycE levels. While the snr1^E1-dependent increased cell growth does not appear to be directly associated with altered expression of G₁ or G₂ cyclins, transcription of the G₂-M regulator string/cdc25 is reduced. Thus, in addition to important functions of the Brm complex in G₁-S control, the complex also appears to be important for transcription of genes required for cell cycle progression (Zraly, 2004).

The conditional mutant snr1^E1 displays wing patterning defects and increased mitotic growth at both the permissive (18° C) and the restrictive (29° C) temperatures. The mutant phenotypes are sensitive to both temperature of incubation and snr1 gene dosage, indicating that they specifically result from reduced or compromised SNR1 function, rather than from complete disruption of Brm complex activities. In contrast to the use of null alleles that may reduce total complex number by half, snr1^E1 produces a stable protein that is assembled into Brm complexes at both temperatures, thus allowing complexes to form and bind their targets, but then are defective in some other function of the complex. This point is critical for these studies, since there are significantly different effects resulting from complete loss of functional Brm complexes or activities as contrasted with impaired functions that result from the incorporation of defective subunits. To help understand the functional roles of SNR1 within the conserved Brm ATP-dependent chromatin remodeling complex during metazoan development, advantage was taken of these dosage- and temperature-dependent snr1^E1 phenotypes, as well as the brm^K804R dominant-negative, both of which result in the incorporation of defective subunits (Zraly, 2004).

This report shows that snr1 can genetically interact with a subset of genes involved in cell cycle control. In addition, co-immunoprecipitation of DmcycE/CDK2 and the Brm complex indicate that stable complexes could form in vivo, while both GST-pulldown and yeast two-hybrid studies suggest that residues within SNR1 might help mediate or stabilize these contacts. SNR1 is strongly conserved with counterparts in yeast (SNF5) and mammals (INI1). The most conserved portions among SNR1-related proteins occur within the ~200-amino-acid C-terminal region comprising two imperfect repeats and a coiled coil. The repeat regions are important for contacts with a variety of cellular factors, including Drosophila Bicoid, the HOX gene regulators TRX and HRX/MLL, c-MYC as well as the viral-encoded proteins HIV integrase and HPV E1. In addition, yeast SNF5 is involved in direct associations with the GAL4 transcriptional activator. Contacts with conserved features of SNR1 are important for recruiting or modulating Drosophila Brm complex functions in vivo. The SNR1/DmCDK2 interaction may also be an important conserved feature, since similar contacts are observed between SNR1 C-terminal residues and mammalian CDK2 using yeast two-hybrid assays (Zraly, 2004).

Components of the mammalian Brm complexes, including the hBrm/BRG-1 and BAF155 (MOR) subunits, are phosphorylated prior to the onset of mitosis and this modification may be important for restricting or modulating complex activity. However, the cell cycle kinase involved and specific target residues within Brm complex components have not been identified. On the basis of work from cultured mammalian cells and the results reported in this study, CycE/CDK2 appears to be among the likely candidates for important regulatory kinase functions during portions of the cell cycle (Zraly, 2004).

CDK2 is capable of forming contacts with SNR1 through the Repeat 2 and coiled-coil regions. What might be the importance of the SNR1-CDK2 interaction? SNR1 and INI1 do not contain any obvious CDK2 phosphorylation sites and SNR1 does not appear to be a phosphoprotein, since assays using a variety of general protein phosphatases produce no detectable change in SNR1 electrophoretic migration on SDS-PAGE gels. This may be misleading, since other putative phosphoproteins, including Drosophila RBF, do not change electrophoretic mobility when treated with phosphatases. However, yeast SFH1p found in the SWI/SNF-related RSC complex and a close relative of SNR1/INI1/SNF5 appears to be phosphorylated during G₁ phase. Thus, while SNR1 does not appear to be the likely direct target for DmcycE/CDK2 regulation, the genetic results suggest the possibility that contacts between SNR1 and CDK2 may serve to stabilize or regulate interactions between DmcycE/CDK2 and the Brm complex or help to direct kinase activity, targeted either to other components of the Brm complex or to unknown cellular proteins (Zraly, 2004).

How might interactions between the Brm chromatin remodeling complex and DmcycE/CDK2 contribute to appropriate cell cycle regulation? A growing body of evidence strongly suggests that ATP-dependent chromatin remodeling complexes perform essential functions in controlling normal mitotic cell cycles. For example, the SWI/SNF complex is important for the expression of mitotic genes and DNA replication in yeast. In mammals, the Brm-related complexes functionally interact with histone deacetylases and pRB to block entry into S phase. As a consequence of losing or misregulating chromatin remodeling activities, normal cell cycle control is disrupted. Specifically, loss of INI1 is associated with aggressive cancers, leads to the rapid development of tumors in knockout mice, and results in G₁-specific defects. Further, overexpression of Cyclin E can abrogate cell cycle arrest caused by the introduction of BRG1 into SW13 adenocarcinoma cells (Zraly, 2004).

The requirements for ATP-dependent chromatin remodeling activities during the cell cycle are likely to be quite complex, perhaps involving known functions in controlling gene transcription (activation and repression) and/or regulating aspects of chromosome replication. In cultured mammalian cells, INI1 was shown to repress cyclinD1 transcription in G₁ phase through collaboration with HDAC1. Unlike mammalian cyclinD, Drosophila DmcycD is not required for entry into S phase, but has been proposed to function during G₁ to regulate cell growth. While snr1^E1 mutant phenotypes are sensitive to Cyclin D levels, the expression of DmcycD is unaffected in the mutant, consistent with the view that the snr1^E1 growth defects are likely due to misregulation of genes downstream of DmcycE, possibly involving targets of E2F regulation (Zraly, 2004).

In addition to demonstrating Brm complex regulation of gene expression during the S and G₂ phases, these results also suggest RNA PolII-independent roles in restricting S-phase entry. For example, SNR1 is excluded from mitotic chromatin during the early embryonic nuclear divisions in the absence of zygotic transcription or G₁-G₂ phases. During these early divisions, type II DmcycE is a potent inducer of S phase and this form exhibits strong in vivo associations with SNR1. One scenario is that the Brm complex is recruited to specific chromosomal sites by sequence-specific repressors where the complex might act to stabilize binding of the repressor and/or remodel nucleosomes in an ATP-dependent manner, thereby establishing a repressive environment to restrict replication initiation. The cellular proteins involved in potentially recruiting the Brm complex to specific loci involved in replication initiation are not presently known, but may include transcription factors, such as RBF/E2F or ORC. Recruitment of CycE/CDK2 to replication origins and interaction with SNR1 might then allow for inactivation of Brm activity and release of the complex from chromatin through phosphorylation of specific subunits. The SNR1^E1 mutant protein likely compromises one or more of these interactions, reducing the effective recruitment of the Brm complex to targets that are normally repressed by Brm complex activities. This could possibly lead to compromised S-phase restriction, partly relieving the requirement for DmcycE/CDK2 activity to allow progression into S phase (Zraly, 2004).

The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting

The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. This study found that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (TIP60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. These results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions (Tea, 2011).

The stereotyped organization of the Drosophila olfactory system makes it an attractive model to study wiring specificity. The first olfactory processing center is the antennal lobe, a bilaterally symmetric structure at the anterior of the Drosophila brain. It is composed of approximately 50 glomeruli in a three-dimensional organization. Each olfactory projection neuron (PN) targets its dendrites to one of those glomeruli to make synaptic connections with a specific class of olfactory receptor neurons. Each PN sends its axon stereotypically to higher brain centers (Tea, 2011).

During development, the dendrites of PNs pattern the antennal lobe prior to axons of olfactory receptor neurons. The specificity of PN dendrite targeting is largely genetically pre-determined by the cell-autonomous action of transcription factors, several of which have been previously described. Furthermore, chromatin remodeling factors have been shown to play an important role in PN wiring (Tea, 2010), although very little is currently known about their specific functions. This study reports a genetic screen for additional factors that regulate PN dendrite wiring specificity; Brahma associated protein 55 kD (Bap55) was identified as a regulator of PN dendrite wiring specificity as part of the TIP60 chromatin remodeling complex (Tea, 2011).

Bap55 is an actin-related protein, the majority of which physically associates with the Brahma (BRM) chromatin remodeling complex in Drosophila embryo extracts. There are two distinct BRM complexes: BAP (Brahma associated proteins; homologous to yeast SWI/SNF) and PBAP (Polybromo-associated BAP; homologous to yeast RSC), both of which contain Brahma, Bap55, and Snf5-Related 1 (Snr1). The human homologs of the BAP and PBAP complexes are called the BAF (Brg1 associated factors) and PBAF (Polybromo-associated BAF) complexes, respectively. The BRM/BAF complexes are members of the SWI/SNF family of ATP-dependent chromatin-remodeling complexes, and have been shown to both activate and repress gene transcription, in some cases, of the same gene (Tea, 2011).

In experiments purifying proteins in complex with tagged Drosophila Pontin in S2 cells, Bap55 was also co-purified as a part of the TIP60 complex, as determined by mass spectrometry. The TIP60 histone acetyltransferase complex has been shown to be involved in many processes, including both transcriptional activation and repression. The complex contains many components, including Bap55, Domino (Dom), and Enhancer of Polycomb (E(Pc)). Dom, homologous to human p400, is the catalytic DNA-dependent ATPase; its ATPase domain is highly similar to Drosophila Brahma and human BRG1 ATPase domains. E(Pc) is homologous to human EPC1 and EPC2 and is an unusual member of the Polycomb group; it does not exhibit homeotic transformations on its own, but rather enhances mutations in other Polycomb group genes (Tea, 2011).

This study provides evidence that Bap55 functions as a part of the TIP60 complex rather than the BRM complex in postmitotic PNs to control their dendrite wiring specificity (Tea, 2011).

To further understanding of dendrite wiring specificity in Drosophila olfactory PNs, a MARCM-based forward genetic screen was performed using piggyBac insertional mutants. MARCM allows visualization and genetic manipulation of single cell or neuroblast clones in an otherwise heterozygous background, permitting the study of essential genes in mosaic animals. In this screen, GH146-GAL4 was used to label a single PN born soon after larval hatching, which in wild-type (WT) animals always projects its dendrites to the dorsolateral glomerulus DL1 in the posterior of the antennal lobe. The DL1 PN also exhibits a stereotyped axon projection, forming an L-shaped pattern in the lateral horn, with additional branches in the mushroom body calyx. A mutant, called LL05955, was identified in which DL1 PNs mistargeted to the dorsolateral glomerulus DA4l in the anterior of the antennal lobe. This phenotype is strikingly specific, with 100% penetrance. Arborization of mutant axons, however, was not obviously altered. The piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. LL05955 is inserted into the coding sequence of Bap55, encoding a homolog of human BAF53a and BAF53b. Precise excision of the piggyBac insertion reverted the dendrite mistargeting phenotype, confirming that disruption of the Bap55 gene causes the dendrite mistargeting (Tea, 2011).

In addition to causing DL1 mistargeting, Bap55 mutants also display neuroblast clone phenotypes. In WT, GH146-GAL4 can label three distinct types of PN neuroblast clones generated in newly hatched larvae. Two of these clones, the anterodorsal neuroblast clone and the lateral neuroblast clone, possess cell bodies that lie dorsal or lateral to the antennal lobe, respectively. PNs from these two lineages project their dendrites to stereotyped and nonoverlapping subsets of glomeruli in the antennal lobe. The third type of clone, the ventral neuroblast clone, has cell bodies that lie ventral to the antennal lobe and dendrites that target throughout the antennal lobe due to the inclusion of at least one PN that elaborates its dendrites to all glomeruli (Tea, 2011).

In Bap55^-/- PNs, anterodorsal neuroblast clones display a mild reduction in cell number, and their dendrites are abnormally clustered in the anterior dorsal region of the antennal lobe, including the DA4l glomerulus. Lateral neuroblast clones display a severe reduction in cell number, and the remaining dendrites are unable to target to many glomeruli throughout the antennal lobe. Ventral neuroblast clones display a mild reduction in cell number and a reduced dendrite mass throughout the antennal lobe. During development, the lateral neuroblast first gives rise to local interneurons before switching to produce PNs; in mutants affecting cell proliferation, this property of the lateral neuroblast displays as a severe reduction in GH146-labeled PNs. The severely reduced cell number in Bap55 mutants suggests that Bap55 is essential for neuroblast proliferation or neuronal survival. In the anterodorsal and ventral neuroblasts, PN numbers are not drastically changed; thus, the phenotypes indicate that Bap55 is important for dendrite targeting in multiple classes of PNs (Tea, 2011).

In WT, Mz19-GAL4 labels a subset of the GH146-GAL4 labeling pattern. It labels a small number of PNs derived from two neuroblasts, which can be clearly identified in WT clones generated in newly hatched larvae. Anterodorsal neuroblast clones target their dendrites to the VA1d glomerulus, as well as the DC3 glomerulus residing immediately posterior to VA1d (not easily visible in confocal stacks). Lateral neuroblast clones target all dendrites to the DA1 glomerulus. Unlike GH146-GAL4, WT Mz19-GAL4 never labels ventral neuroblast clones because it is not normally expressed in those cells (Tea, 2011).

In Bap55 mutant PN clones, however, Mz19-GAL4 labels additional PNs in anterodorsal, lateral, and ventral clones compared to their WT counterparts. This phenotype suggests that some Mz19-negative PNs now express Mz19-GAL4. In anterodorsal clones, Mz19-GAL4 labels additional cells, although not as many as are labeled by GH146-GAL4. The PNs also mistarget their dendrites to the anterior antennal lobe, similar to mutant GH146-GAL4 anterodorsal neuroblast clones. WT lateral neuroblast clones normally contain GH146-positive PNs and GH146-negative local interneurons. In Bap55^-/- lateral neuroblast clones, Mz19-GAL4 predominantly labels local interneurons that send their processes to many glomeruli throughout the antennal lobe and do not send axon projections to higher brain centers. Lateral clones also show ectopic PN labeling with a lower frequency. The Bap55 mutant appears to cause derepression of Mz19-GAL4, resulting in labeled local interneurons. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in Bap55 mutants. This further indicates a derepression of the Mz19-GAL4 labeling pattern (Tea, 2011).

Unlike GH146-GAL4, WT Mz19-GAL4 never labels single cell clones when clone induction is performed in newly hatched larvae. This is because Mz19-GAL4 is not expressed in the DL1 PN, the only GH146-positive cell generated during this heat shock time of clone generation. However, in Bap55 mutant PN clones, Mz19-GAL4 ectopically labels single cell anterodorsal PN clones targeting to the DA4l glomerulus, which show an L-shaped pattern in the lateral horn with branches in the mushroom body calyx, similar to GH146-GAL4 labeling. The simplest interpretation is that this compound phenotype reflects first a derepression of Mz19-GAL4 in the DL1 PN, and second a DL1 to DA4l mistargeting phenotype in Bap55 mutants (Tea, 2011).

To test whether Bap55 functions in the neuroblast or postmitotically in PNs, GH146-GAL4, which expresses only in postmitotic PNs, was used to express UAS-Bap55 in a Bap55^-/- single cell clone. The dendrite mistargeting phenotype was shown to be rescued to the WT DL1 glomerulus and it is concluded that Bap55 functions postmitotically to regulate PN dendrite targeting. The axon phenotype remains the stereotypical L-shaped pattern (Tea, 2011).

The Drosophila Bap55 protein is 70% similar and 54% identical to human BAF53a and 71% similar and 55% identical to human BAF53b. BAF53a and b are 91% similar and 84% identical to each other. Using GH146-GAL4 to express human BAF53a or b in a Bap55^-/- single cell clone, it was found that the human homologs can effectively rescue the Bap55^-/- dendrite mistargeting phenotype. Interestingly, both also cause the de novo DM6 dendrite and ventral axon mistargeting phenotypes in 6 out of 19 cases for BAF53a and 2 out of 32 cases for BAF53b. Thus, human BAF53a and b can largely replace the function of Drosophila Bap55 in PNs (Tea, 2011).

To address whether Bap55 functions as a part of the BRM complex in PN dendrite targeting, two additional BRM complex mutants were tested for their PN dendrite phenotypes. First, Brahma (brm), the catalytic ATPase subunit of the BRM complex, which is required for the activation of many homeotic genes in Drosophila, was tested. Null mutations have been shown to decrease cell viability and cause peripheral nervous system defects. RNA interference knockdown of brm in embryonic class I da neurons revealed dendrite misrouting phenotypes, although not identical to the Bap55 phenotype. The human homologs of brm, BRM and BRG1 (Brahma-related gene-1), both have DNA-dependent ATPase activity. Inactivation of BRM in mice does not yield obvious neural phenotypes, but inactivation of BRG1 in neural progenitors results in reduced proliferation. BRG1 is likely to be required for various aspects of neural development, including proper neural tube development (Tea, 2011).

In PNs, brm mutants displayed anterodorsal single cell clone mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone differing from the next. This is in contrast to the highly stereotyped DA4l mistargeting of Bap55 mutants. brm^-/- neuroblast clones also displayed phenotypes where dendrites make small, meandering projections throughout the antennal lobe, which does not resemble the Bap55^-/- phenotype. They additionally exhibit a perturbed cell morphology phenotype, which is markedly more severe than the Bap55 mutant phenotype (Tea, 2011).

Next, Snr1, a highly conserved subunit of the BRM complex, was tested. It is required to restrict BRM complex activity during the development of wing vein and intervein cells and functions as a growth regulator. Its human homolog, SNF5, is strongly correlated with many cancers, yet little is known about its specific role in neurons (Tea, 2011).

In PNs, Snr1 mutants displayed similar phenotypes to brm mutants. The single cell clones displayed mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone demonstrating a unique phenotype. The neuroblast clones exhibited small meandering dendrites throughout the antennal lobe, which also showed extremely perturbed cell morphology. These results, especially the non-sterotyped single cell clone phenotypes, indicate that the BRM complex functions differently from Bap55 in controlling PN dendrite targeting (Tea, 2011).

brm and Snr1 mutants were further analyzed with Mz19-GAL4 to determine whether their phenotypes resembled the Bap55 mutant phenotype of derepression. It was not possible to generate any labeled clones, indicating one of three possibilities: increased cell death, severe cell proliferation defects, or repression of the Mz19-GAL4 labeling pattern. In any of the three cases, the phenotype does not resemble the Bap55^-/- mutant phenotype of abnormal activation of Mz19-GAL4 in single cell or neuroblast clones, indicating that the BRM complex functions differently from Bap55 in PNs (Tea, 2011).

In the same screen in which the Bap55 mutation was identified, LL05537, a mutation in dom that resulted in a qualitatively similar phenotype to Bap55 mutants was identified. dom^-/- DL1 PNs split their dendrites between the posterior glomerulus DL1 and the anterior glomerulus DA4l. Their axons exhibit a WT L-shaped pattern in the lateral horn (Tea, 2011).

The LL05537 allele contains a piggyBac insertion in an intron just upstream of the translation start of dom. Because the piggyBac insertion contains splice acceptor sites and stop codons in all three coding frames, this allele likely disrupts all isoforms of dom. Similarly to Bap55, the piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. Precise excision of the piggyBac insertion reverted the dendrite targeting phenotype, confirming that disruption of the dom gene causes the dendrite mistargeting. In addition, a BAC transgene that contains the entire dom transcriptional unit rescued the dom^-/- mutant phenotypes (Tea, 2011).

Dom is the catalytic DNA-dependent ATPase of the TIP60 complex and has been shown to contribute to a repressive chromatin structure and silencing of homeotic genes. Dom is a member of the SWI/SNF family and its ATPase domain is highly similar to the Drosophila Brahma and human BRG1 ATPase domains. The human homolog of Dom is p400, which is important for regulating nucleosome stability during repair of double-stranded DNA breaks and an important regulator of embryonic stem cell identity (Tea, 2011).

To determine whether Bap55 and Dom genetically interact, UAS-Bap55 was expressed in a dom^-/- PN. This manipulation did not significantly alter the dendrite phenotype. The axon branching pattern also was not altered (Tea, 2011).

Another component of the TIP60 complex, E(Pc), was also examined. In Drosophila, E(Pc) is a suppressor of position-effect variegation and heterozygous mutations in E(Pc) result in an increase in homologous recombination over nonhomologous end joining at double-stranded DNA breaks. Following ionizing radiation, heterozygous animals also exhibit higher genome stability and lower incidence of apoptosis. Yet little is known about its role in neurons (Tea, 2011).

In this study, it was found that E(Pc)^-/- DL1 PN dendrites also mistarget to the anterior glomerulus DA4l and exhibit the stereotyped L-shaped axon pattern in the lateral horn. A BAC transgene that contains the entire E(Pc) transcription unit rescued the E(Pc) mutant phenotypes. To determine whether Bap55 and E(Pc) genetically interact, UAS-Bap55 was expressed in an E(Pc)^-/- DL1 PN. This manipulation caused the dendrites to split between the DA4l and DM6 glomeruli, and resulted in axons targeting ventrally to the lateral horn (Tea, 2011).

Neuroblast clones mutant for dom also exhibit dendrite mistargeting phenotypes to inappropriate glomeruli throughout the antennal lobe. Anterodorsal and lateral neuroblast clones show a very mild reduction in cell number and their dendrites do not target to the full set of proper glomeruli. Ventral neuroblast clones, when compared to WT, exhibit incomplete targeting throughout the antennal lobe (Tea, 2011).

Further analysis of dom mutants by labeling with Mz19-GAL4 revealed the same derepression as in Bap55 mutants. dom mutant Mz19-GAL4 PN clones also label anterodorsal, lateral, and ventral neuroblast clones with phenotypes similar to GH146-GAL4 labeled neuroblast clones. In anterodorsal and lateral neuroblast clones, Mz19-GAL4 labels a large number of PNs that target to many glomeruli throughout the antennal lobe, although the cell number is smaller than GH146-GAL4 labeling. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in dom mutants. Mz19-GAL4 also labels single cell clones that split their dendrites between the DA4l and DL1 glomeruli and form the stereotypical L-shaped axon pattern in the lateral horn. As in Bap55 mutants, this compound phenotype likely results from ectopic labeling of a DL1 PN, which further mistargets to DA4l (Tea, 2011).

The E(Pc) phenotypes in GH146 and Mz19-GAL4 labeled neuroblast clones, as well as Mz19-GAL4 labeled single cell clones, displayed similar phenotypes to dom as described above. The phenotypic similarities in single cell clone dendrite mistargeting and derepression of a PN-GAL4 in mutations that disrupt Bap55, dom and E(Pc) strongly suggest that these three proteins act together in the TIP60 complex to regulate PN development (Tea, 2011).

This study has demonstrated a similar role for three members of the TIP60 complex in olfactory PN wiring. The TIP60 complex plays a very specific role in controlling dendrite wiring specificity, with a precise mistargeting of the dendrite mass in Bap55, dom, and E(Pc) mutants. This specific DL1 to DA4l mistargeting phenotype has only been seen in these three mutants, out of approximately 4,000 other insertional and EMS mutants screened, supporting the conclusion that the TIP60 complex has a specific function in controlling PN dendrite targeting. TIP60 complex mutants show discrete glomerular mistargeting, rather than randomly distributed dendrite spillover to different glomeruli. In contrast, perturbation of individual cell surface receptors often leads to variable mistargeted dendrites that do not necessarily obey glomerular borders, possibly reflecting the combinatorial use of many cell surface effector molecules. Even transcription factor mutants yield variable phenotypes. Interestingly, BRM complex mutants yield non-stereotyped phenotypes in PNs. No stereotyped glomerular targeting was seen for brm or Snr1 mutant dendrites; each PN spreads its dendrites across different glomeruli. These data suggest that different chromatin remodeling complexes play distinct roles in regulating neuronal differentiation. The uni- or bi-glomerular targeting to specific glomeruli implies that the TIP60 complex sits at the top of a regulatory hierarchy to orchestrate an entire transcriptional program of regulation (Tea, 2011).

This study suggests a function for Bap55 in Drosophila olfactory PN development as a part of the TIP60 complex rather than the BRM complex. Another possibility could be that Bap55 also serves as the interface between the BRM and TIP60 complexes. While loss of core BRM complex components results in a more general defect, loss of Bap55 could specifically disrupt interactions with the TIP60 complex but maintain other BRM complex functions, causing a more specific targeting phenotype mimicking loss of TIP60 complex components (Tea, 2011).

Interestingly, both human BAF53a and b can significantly rescue the Bap55^-/- phenotype. Though in mammals BAF53a is expressed in neural progenitors and BAF53b is expressed in postmitotic neurons, they can perform the same postmitotic function in Drosophila PNs. Further, both can function with the TIP60 complex in PNs to regulate wiring specificity. These data suggest that the functions for BAF53a and b (in neural precursors and postmitotic neurons, respectively) diverge after the evolutionary split between vertebrates and insects (Tea, 2011).

The discrete glomerular states of the mistargeting phenotypes may suggest a role for the TIP60 complex upstream of a regulatory hierarchy determining PN targeting decisions. It is possible that disrupting various components changes the composition of the complex. Additionally, overexpression of Bap55 in various mutant backgrounds might alter the sensitive stoichiometry of the TIP60 complex, resulting in targeting to different but still distinct glomeruli (Tea, 2011).

Several mutants have been identified that cause DL1 PNs to mistarget to areas near the DM6 glomerulus (Tea, 2010). Interestingly, WT DM6 PNs have the most similar lateral horn axon arborization pattern to DL1 PNs. It is hypothesized that the transcriptional code for DM6 is similar to that of DL1, which is at least partially regulated by the TIP60 complex. The genes described in this manuscript are the only mutants that have yielded specific DA4l mistargeting to date. It is possible that the targeting 'code' for DA4l, DL1, and DM6 may be most similar, such that perturbation of the TIP60 complex might result in reprogramming of dendrite targeting. PNs have previously been shown to be pre-specified by birth order. Yet DA4l is born in early embryogenesis, DL1 is born in early larva, and DM6 is born in late larva. This implies that the TIP60 transcriptional code does not correlate with PN birth order. The mechanisms by which the TIP60 complex specifies PN dendrite targeting remain to be determined (Tea, 2011).

This study has characterize PN phenotypes of mutants in the BRM and TIP60 complexes, with a focus on Bap55, which is shared by the two complexes. The TIP60 complex was found to play a very specific role in regulating PN dendrite targeting; mutants mistarget from the DL1 to the DA4l glomerulus. This specific mistargeting phenotype suggests that TIP60 controls a transcriptional program important for making dendrite targeting decisions (Tea, 2011).

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 (http://www.flyatlas.org/) 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).

Histone lysine demethylases function as co-repressors of SWI/SNF remodeling activities during Drosophila wing development

The conserved SWI/SNF chromatin remodeling complex uses the energy from ATP hydrolysis to alter local chromatin environments through disrupting DNA-histone contacts. These alterations influence transcription activation, as well as repression. The Drosophila SWI/SNF counterpart, known as the Brahma or Brm complex, has been shown to have an essential role in regulating the proper expression of many developmentally important genes, including those required for eye and wing tissue morphogenesis. A temperature sensitive mutation in one of the core complex subunits, SNR1 (SNF5/INI1/SMARCB1), results in reproducible wing patterning phenotypes that can be dominantly enhanced and suppressed by extragenic mutations. SNR1 functions as a regulatory subunit to modulate chromatin remodeling activities of the Brahma complex on target genes, including both activation and repression. To help identify gene targets and cofactors of the Brahma complex, advantage was taken of the weak dominant nature of the snr1^E1 mutation to carry out an unbiased genetic modifier screen. Using a set of overlapping chromosomal deficiencies that removed the majority of the Drosophila genome, genes were sought that when heterozygous would function to either enhance or suppress the snr1^E1 wing pattern phenotype. Among potential targets of the Brahma complex, components were identified of the Notch, EGFR and DPP signaling pathways important for wing development. Mutations in genes encoding histone demethylase enzymes were identified as cofactors of Brahma complex function. In addition, it was found that the Lysine Specific Demethylase 1 gene (lsd1) was important for the proper cell type-specific development of wing patterning (Curtis, 2011).

Although chromatin remodeling is an important component of gene activation, its role in gene repression is not as well understood. The unbiased genetic screen using a weak dominant temperature sensitive mutant allele of a key Brm complex regulatory subunit has provided new insights into the involvement of chromatin remodeling complexes in developmental tissue patterning. Mutations in components of several signaling pathways, including Notch, EGFR and DPP/TGFβ, genetically interacted in these assay. These results, combined with candidate gene genetic analyses, have confirmed a previous hypotheses that the Brm complex participates in both gene activation and gene repression to help coordinate several key signaling pathways that lead to proper animal patterning. The results are largely concordant with the results of previous limited screens that identified a set of dominant modifiers of brm^K804R mutant phenotypes. Among 14 chromosomal deficiencies that enhanced the brm^K804R rough eye phenotype, this study found that 6/14 were also dominant enhancers of the snr1^E1 wing phenotype and 3/14 were suppressors, suggesting that dominant modifier screens are effective tools for identifying unknown loci important for Brm complex regulatory functions. Consistent with this view, the Brm complex has been shown to interact the Notch ligand, Delta, in the developing fly eye. The genetic modifier screen results presented in this study indicate that Notch signaling functions may also be mediated through the Brm complex in the developing fly wing. Given the strong evolutionary conservation of these pathways, it is anticipated that the vertebrate SWI/SNF orthologs will play a similarly important role in patterning the tissues of vertebrate animals (Curtis, 2011).

What are the target genes regulated by the Brm complex in the developing wing? Previous studies have found that loss of snr1 function results in ectopic dpp and rhomboid expression in intervein cells. These data are consistent with the genetic interactions shown in this report that were observed using mutants affecting both the DPP and EGFR pathways. These studies have additionally provided an important insight into gene regulatory factors beyond signaling pathways that contribute to transcription repression in collaboration with chromatin remodeling complexes at key points in the development and differentiation of tissues. In the present analyses, several lines of evidence are provided suggesting that the mechanism of Brm complex-mediated gene repression is not only dependent upon a tight, physical and genetic relationship between two core subunits, SNR1 and MOR, but also on histone lysine demethylase enzymes (Curtis, 2011).

It has been reported that the full in vitro chromatin remodeling activity of the mammalian BRM/BRG1 complex on reconstituted nucleosomes can be accomplished with a subset of three or four core components, including the SNF5 (SNR1), BAF155/BAF170 (MOR) and BRM/BRG1 ATPase subunits that are highly conserved from yeast to vertebrates. Each of these subunits is required for complex stability in vivo as RNAi depletion of the individual components in cultured Drosophila cells leads to reduced stability of the other subunits with corresponding changes in target gene expression. Loss of BRM function in vivo, using either a dominant negative ATPase deficient mutant (brm^K804R) or an amorphic allele (brm²), can suppress the snr1^E1 wing phenotype revealing an important role for SNR1 in restraining Brm complex transcription activation functions. In contrast, mor mutants enhance mutant phenotypes associated with reduced brm function and show allele-specific interaction with snr1^E1, suggesting an important functional relationship between the MOR, BRM and SNR1 subunits. MOR likely serves as a scaffolding protein, since physical associations were observed between SNR1-MOR and MOR-BRM. Two independent domains of MOR, the SWIRM and SANT, domains respectively, are critical for the binding interaction. Therefore, the contribution of SNR1 regulatory function on Brm complex chromatin remodeling activities may depend on crosstalk through MOR since no direct physical contacts between SNR1 and the BRM subunit have been observed (Curtis, 2011).

An unbiased dominant modifier genetic screen allowed identification of histone lysine demethylase enzymes as novel coregulators of the Brm complex in controlling gene expression. Previous screens looking for modifiers of a brm dominant negative allele (brm^K804R) did not uncover mutations in histone-modifying families, such as acetyltransferases, deacetylases, and methyltransferases. However, the wing patterning defect associated with snr1^E1 is highly sensitive, allowing observation of subtle changes in remodeling activities, and identification a family of epigenetic modifiers as potential Brm regulators. Previous studies have found that histone deacetylases (HDACs) were important corepressors that worked in direct collaboration with the Brm complex. In the present study, mutations in predicted demethylase genes genetically interacted with snr1^E1 and LSD1 was shown to associate with the Brm complex in vivo, suggesting demethylases are also potential cofactors. While a functional cooperation between histone deacetylation and demethylation activities has been suggested previously, the current data implicates at least three chromatin modifying activities—ATP-dependent chromatin remodeling, histone deacetylation and demethylation—cooperating to regulate tissue-specific gene repression through multiple bridging interactions. In this scenario, the commitment of a gene promoter to be repressed in a cell type-specific manner would depend on the collateral influence of several chromatin modifying activities that would serve to help establish a repressed transcriptional environment, refractory to the influence of signaling pathways operational in adjacent cells (Curtis, 2011).

There appears to be no correlation between the predicted demethylase lysine substrate and enhancement/suppression of the snr1^E1 phenotype. This is not surprising, since a high degree of functional redundancy exists amongst demethylase enzymes. It is likely that multiple demethylase enzymes cooperate to regulate a variety of target genes. This is supported by experimental evidence showing that knockdown experiments of individual demethylases, for example lsd1, in cell culture often showed little or no change in global methylation status, though significant changes were observed on a gene-specific level in vivo. Independent loss of function mutations in two JARID family members, lid and Jarid2/CG3654, resulted in an opposite genetic interaction with snr1^E1. This study observed that a loss of function mutation in lid, (lid²) dominantly suppressed, whereas a loss of function mutation in Jarid2 (CG3654^EY02717) enhanced the ectopic vein phenotype associated with snr1^E1. LID is an H3K4me3/me2 specific demethylase. JARID2 is predicted to have the same substrate specificity, though overexpression analyses in cell culture experiments showed no global increase in H3K4me3/2. The observed opposite genetic interaction with snr1^E1 may reflect differences in target gene regulation by LID and JARID2, either as a consequence of different target genes controlled in the developing wing or through opposite mechanisms in controlling gene transcription. Importantly, JARID2 homologs in Xenopus and mammalian model systems physically associate with the Polycomb Repressor Complex-2 (PRC2) and directly contribute to transcriptional repression by preventing the methylation of the histone lysine residues correlated with transcriptional activation. Therefore, mutation of JARID2 (CG3654^EY02717) may enhance the snr1^E1 phenotype if the normal role of CG3654 is to suppress transcription of a particular gene involved in wing vein development (Curtis, 2011).

The cell-fate decision to become vein or intervein is largely based on cell-type specific expression of transcription factors. In vein cells, transcription factors with gene targets that promote vein development are highly expressed, whereas those with gene targets that block vein fate are repressed. In intervein cells, the opposite is observed, with heightened expression intervein-promoting factors and decreased expression of vein promoting factors. The Brm complex has an important role in development of both cell fates, serving a positive role to promote vein development in vein cells, and repress vein development in intervein cells. The opposite genetic interaction phenotypes observed with lid and Jarid2 could be partially explained if the Brm complex is coordinating with the each specific demethylase to regulate different target genes. This study found that loss of function mutations in vein promoting genes, such as Egfr, suppressed the snr1^E1 phenotype. The results suggest that LID and EGFR may regulate the expression of similar target genes and indeed EGFR (as well as other signaling pathways) may function in wing vein development through LID. In this scenario, a loss of function mutation in lid would result in a decrease in the expression of vein promoting genes, thereby suppressing the snr1^E1 ectopic vein phenotype. Enhancement of the snr1^E1 phenotype by Jarid2/CG3654^EY02717 can be explained if JARID2 promotes activation of genes required to block vein differentiation, just as loss of function mutations in vein-inhibiting factors, such as net, enhanced the snr1^E1 phenotype (Curtis, 2011).

The candidate genetic screen results suggest that histone lysine demethylase enzymes are likely cofactors of Brm chromatin remodeling activity. However, it is highly unlikely that stable physical associations are made between the complex and all six demethylases. The possibility cannot be eliminated that the Brm complex and demethylase enzymes are independently regulating genes involved in wing patterning or eliciting their functions on different targets at different times during development to contribute to the final read-out of vein/intervein patterning in the adult wing. However, a direct physical association was detected between the Brm complex and LSD1 in coimmunoprecipitation and GST-pulldown experiments, implying that LSD1 is a potential cofactor of Brm complex remodeling activities (Curtis, 2011).

The genetic epistasis experiments demonstrated an important in vivo functional relationship between LSD1 and the core subunits of the Brm complex, SNR1, MOR, and BRM. Brm complexes can be subdivided into two groups: PBAP complexes contain BAP170, POLYBROMO/BAP180, and SAYP, whereas BAP complexes contain OSA. These complexes can regulate target genes in a synergistic, antagonistic, or independent manner. BAP and PBAP complexes likely have differential regulatory functions, since they have distinct, but overlapping, localization patterns on larval salivary gland polytene chromosomes and targeted knockdown of OSA, POLYBROMO, or BAP180 using RNAi in cultured Schneider cells, leads to differential expression profiles on whole genome arrays. OSA, BAP170, BAP180, and SAYP likely have different roles in development, as mutation of each leads to different abnormalities. For example, BAP180 is required for proper egg shell development, whereas BAP170 is necessary to stabilize BAP180, important for adult viability, and vein cell differentiation. OSA is necessary for photoreceptor development, normal embryonic segmentation, and wing patterning. BAP, but not PBAP complexes have an important role in regulating cell cycle progression through mitosis (Curtis, 2011).

In mice, knockout of Baf180 causes misregulation of retinoic acid receptor target genes and heart developmental defects, indicating that PBAP complexes may have a role in nuclear receptor transcriptional regulation. The LSD1 corepressor complex, including the cofactor proteins, CoREST (see Drosophila CoREST), and histone deacetylase, HDAC1/2, have also been indicated in nuclear receptor transcriptional regulation. LSD1 association in complexes containing the Estrogen Receptor (ER) or Androgen Receptor (AR) leads to a switch in methylated lysine specificity, and results in demethylation of mono- and dimethylated H3K9 and gene activation (Curtis, 2011).

It is not known how BAP vs. PBAP complexes are differentially recruited to target genes. Recruitment of BAP complexes to specific target genes may depend on the physical associations made by OSA and sequence-specific transcription factors. For example, OSA is required for expression of target genes associated with the transcription factors Pannier and Apterous and can promote transcriptional repression of genes regulated by Wnt/Wingless signaling. Genetic epistasis experiments reveal that LSD1 cooperates with PBAP, but not BAP containing complexes in the Drosophila wing, suggesting that the physical association observed between LSD1 and Brm complex may be limited to PBAP complexes and provide a mechanism for selective target gene recruitment and regulation by Brm remodeling complexes. Further analyses, such as GST-pulldown and coimmunoprecipitation experiments using PBAP specific components need to be performed to address this possibility (Curtis, 2011).

Ectopic vein development within intervein tissue can result from two different possibilities: 1) the loss of a factor necessary to block vein cell development, or 2) the gain of a factor that promotes vein cell differentiation. Knockdown experiments suggest LSD1/dCoREST functions through the first mechanism. Loss of LSD1/dCoREST throughout the entire developing wing imaginal disc resulted in the development of vein material in intervein tissue, but no changes in vein morphology were observed. If LSD1/dCoREST normally functioned to promote vein development, then loss throughout the entire wing should have led to a loss of vein phenotype (Curtis, 2011).

Several lines of evidence suggest that LSD1 may be capable of regulating gene transcription in a cell-type or stage dependent manner. The affect of homozygous loss of lsd1 on transcriptional regulation of known target genes, including the Sodium Channel and NicotinicAcetylcholine Receptor-β is minimal in embryos and larvae, but significant in pupae. This implies that LSD1 has an important role in regulating gene transcription during later developmental stages. Moreover, LSD1 negative regulation of the homeobox genes, Ultrabithorax (Ubx) and abdominal-B (abd-B) continues into adulthood, as lsd1 null animals display significantly increased expression of these genes as the animals continue to age. This stage-dependent requirement appears to be conserved, as the conditional knock-out of LSD1 in the developing mouse pituitary gland causes little or no morphological defects early in pituitary development (E9-9.5), but significantly alters cell-fate determination choices during later stages (E17.5). Furthermore, LSD1 mediates both gene activation and gene repression of different target genes by associating with several multisubunit complexe (Curtis, 2011).

Knockdown and genetic epistasis experiments further support the idea that LSD1 is important for regulating terminal differentiation, since patterning phenotypes are similar to those observed with defects in DPP and EGFR signaling, the pathways active during pupal development, rather than observed with defects in HH signaling, an early pathway component. Previous work has demonstrated an important role in Brm complex involvement in EGFR, DPP, and Delta/N signaling. More recently, it has been demonstrated that OSA, the defining subunit of the BAP complex, is required to activate EGFR targets in the developing wing. In this regard, the Brm complex may be cooperating with LSD1 to regulate several conserved signaling pathways, but this cooperation may be tissue and developmental time-point dependent (Curtis, 2011).

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date revised: 15 February 2013

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