Transcriptional Regulation

Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling

Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. This study shows that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped-Yorkie transcriptional complex, and the Hippo (Hpo) signaling pathway directly restricts ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promotes ISC proliferation. These findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation (Jin, 2013).

SWI/SNF complex subunits regulate the chromatin structure by shutting off or turning on the gene expression during differentiation. Recently, the findings from several research reports based on the stem cell system reveal important roles of chromatin remodeling complex in stem cell state maintenance. The current study suggests that the chromatin remodeling activity of Brm complex is required for the proliferation and differentiation of Drosophila ISCs. Based on these findings, it is proposed that Brm is critical for maintaining Drosophila intestinal homeostasis. High levels of Brm in the ISC nucleus represent high proliferative ability and are essential for EC differentiation; low levels of Brm in the EC nucleus may be a response for homeostasis. Changes in Brm protein levels result in the disruption of differentiation and deregulation of cell proliferation. In line with previous findings in human, the cell-type-specific expression of Drosophila homologs BRG1 and BRM are also detected in adult tissues. BRG1 is mainly expressed in cell types that constantly undergo proliferation or self-renewal, whereas BRM is expressed in other cell types. These observations indicate that Brm may act similarly to BRG1 and BRM in controlling proliferation and differentiation (Jin, 2013).

The Hpo pathway restricts cell proliferation and promotes cell death at least in two ways: inhibiting the transcriptional co-activator Yki and inducing activation of pro-apoptotic genes such as caspases directly. This study has identified a novel regulatory mechanism of the Hpo pathway in maintaining intestinal homeostasis. In this scenario, Brm activity is regulated by the Hpo pathway. In normal physiological conditions, under the control of Hpo signaling, the function of Yki–Sd to promote ISC proliferation is restricted and the pro-proliferation of target genes such as diap1 that inhibits Hpo-induced caspase activity cannot be further activated. Therefore, Hpo signaling normally functions to restrict cell numbers in the midgut by keeping ISC proliferation at low levels. Yki is enriched in ISCs, but predominantly inactivated in cytoplasm by the Hpo pathway. The knockdown of Yki in ISCs did not cause any phenotype in the midgut, suggesting that Yki is inactivated in ISCs under normal homeostasis. During an injury, Hpo signaling is suppressed or disrupted, Yki translocates into the nuclei to form a complex with Sd, which may allow Yki–Sd to interact with Brm complex in the nucleus to activate transcriptional targets. Of note, the loss-of-function of Brm resulted in growth defect of ISCs, suggesting that Brm is required for ISC homeostasis and possessing a different role of Brm from Yki in the regulation of ISCs. It is possible that the function of Brm on ISC homeostasis is regulated via other signaling pathways by recruiting other factors. Therefore, different phenotypes induced by the loss-of-function of Brm and Yki in midgut might be due to different regulatory mechanisms. Despite its unique function cooperating with Yki in midgut, that Brm complex is essential for Yki-mediated transcription might be a general requirement for cell proliferation. While this manuscript was under preparation, Irvine lab reported a genome-wide association of Yki with chromatin and chromatin-remodeling complexes (Oh, 2013). These results support the model developed in this paper (Jin, 2013).

The current results also suggest that the interaction between Brm and Yki–Sd transcriptional complex is under tight regulation. The loss of Hpo signaling stabilizes Brm protein, whereas the active Hpo pathway restricts Brm levels by activating Drosophila caspases to cleave Brm at the D718 site and inhibiting downstream target gene diap1 transcription simultaneously. In addition, overexpression of Brm complex components induces only a mild enhancement on midgut proliferation. One possibility is that overexpressing only one of the Brm complex components does not provide full activation of the whole complex; the other possibility is that due to the restriction of the Hpo signaling, as overexpressing BrmD718A mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm and coexpression of BrmD718A completely rescues the impairment of Hpo-induced ISC proliferation. D718A mutation blocks the caspase-dependent Brm cleavage and exhibits high activity in promoting ISC proliferation. This study has defined a previously unknown, yet essential epigenetic mechanism underlying the role of the Hpo pathway in regulating Brm activity (Jin, 2013).

It is a novel finding that Brm protein level is regulated by the caspase-dependent cleavage. To focus on the function of Brm cleavage in the presence of cell death signals, attempts were made to examine the activities of the cleaved Brm fragments. Although in vivo experiments did not show strong activity of Brm N- and C-cleavage products in promoting proliferation of ISCs, the C-terminal fragment of Brm that contains the ATPase domain exhibits a relative higher activity than the N-terminal fragment in ISCs. The cleavage might induce faster degradation of Brm N- and C-terminus, since it was difficult to detect N- or C-fragments of Brm by Western blot analysis without MG132 treatment. It reveals that the degradation events of Brm including both ubiquitination and cleavage at D718 site can be important for Brm functional regulation under different conditions. To this end, the intrinsic signaling(s) may balance the activity of Brm complex through degradation of some important components, such as Brm, to maintain tissue homeostasis. Of note, the cleavage of Brm at D718 is occurred at a novel DATD sequence that is not conserved in human Brm. It has been reported that Cathepsin G, not caspase, cut hBrm during apoptosis, suggesting that the cleavage regulatory mechanism of Brm is relatively conserved between Drosophila and mammals (Jin, 2013).

This study provides evidence that the Brm complex plays an important role in Drosophila ISC proliferation and differentiation and is regulated by multi-levels of Hpo signaling. The findings indicate that Hpo signaling not only exhibits regulatory roles in organ size control during development but also directly regulates epigenetics through a control of the protein level of epigenetic regulatory component Brm. In mammals, it is known that Hpo signaling and SWI/SNF complex-mediated chromatin remodeling processes play critical roles in tissue development. Malfunction of the Hpo signaling pathway and aberrant expressions of SWI/SNF chromatin-remodeling proteins BRM and BRG1 have been documented in a wide variety of human cancers including colorectal carcinoma. Thus, this study that has implicated a functional link between Hpo signaling pathway and SWI/SNF activity may provide new strategies to develop biomarkers or therapeutic targets (Jin, 2013).

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).

Systematic protein location mapping reveals five principal chromatin types in Drosophila cells

Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, this study shows that the genome is segmented into five principal chromatin types (see Chromatin types are characterized by distinctive protein combinations and histone modifications) that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. A repressive chromatin type was identified that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, evidence is provided that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell (Filion, 2010).

By systematic integration of 53 protein location maps this study found that the Drosophila genome is packaged into a mosaic of five principal chromatin types, each defined by a unique combination of proteins. Extensive evidence demonstrates that the five types differ in a wide range of characteristics besides protein composition, such as biochemical properties, transcriptional activity, histone modifications, replication timing, DNA binding factor (DBF) targeting, as well as sequence properties and functions of the embedded genes. This validates the classification by independent means and provides important insights into the functional properties of the five chromatin types (Filion, 2010).

Identifying five chromatin states out of the binding profiles of 53 proteins comes out as a surprisingly low number (one can form approximately 1016 subsets of 53 elements). It is emphasized that the five chromatin types should be regarded as the major types. Some may be further divided into sub-types, depending on how fine-grained one wishes the classification to be. For example, within each of the transcriptionally active chromatin types, promoters and 3' ends of genes exhibit (mostly quantitative) differences in their protein composition and thus could be regarded as distinct sub-types. However, these local differences are minor relative to the differences between the five principal types that are described in this study. It cannot be excluded that the accumulation of binding profiles of additional proteins would reveal other novel chromatin types. It is also anticipated that the pattern of chromatin types along the genome will vary between cell types. For example, many genes that are embedded in 'BLACK' chromatin (defined in Kc167 cells) are activated in some other cell types. Thus, the chromatin of these genes is likely to switch to an active type (Filion, 2010).

While the integration of data for 53 proteins provides substantial robustness to the classification of chromatin along the genome, a subset of only five marker proteins (histone H1, PC, HP1, MRG15 and BRM), which together occupy 97.6% of the genome, can recapitulate this classification with 85.5% agreement. Assuming that no unknown additional principal chromatin types exist in some cell types, DamID or ChIP of this small set of markers may thus provide an efficient means to examine the distribution of the five chromatin types in various cells and tissues, with acceptable accuracy (Filion, 2010).

Previous work on the expression of integrated reporter genes had suggested that most of the fly genome is transcriptionally repressed, contrasting with the low coverage of PcG and HP1-marked chromatin. BLACK chromatin, which consists of a previously unknown combination of proteins and covers about half of the genome, may account for these observations. Essentially all genes in BLACK chromatin exhibit extremely low expression levels, and transgenes inserted in BLACK chromatin are frequently silenced, indicating that BLACK chromatin constitutes a strongly repressive environment. Importantly, BLACK chromatin is depleted of PcG proteins, HP1, SU(VAR)3-9 and associated proteins, and is also the latest to replicate, underscoring that it is different from previously characterized types of heterochromatin (identified as BLUE and GREEN chromatin in this study) (Filion, 2010).

The proteins that mark BLACK domains provide important clues to the molecular biology of this type of chromatin. Loss of Lamin (LAM), Effete (EFF) or histone H1 causes lethality during Drosophila development. Extensive in vitro and in vivo evidence has suggested a role for H1 in gene repression, most likely through stabilization of nucleosome positions. The enrichment of LAM points to a role of the nuclear lamina in gene regulation in BLACK chromatin, consistent with the long-standing notion that peripheral chromatin is silent. Depletion of LAM causes derepression of several LAM-associated genes (Shevelyov, 2009), while artificial targeting of genes to the nuclear lamina can reduce their expression, suggesting a direct repressive contribution of the nuclear lamina in BLACK chromatin. D1 is a little-studied protein with 11 AT-hook domains. Overexpression of D1 causes ectopic pairing of intercalary heterochromatin (Smith, 2010), suggesting a role in the regulation of higher-order chromatin structure. SUUR specifically regulates late replication on polytene chromosomes (Zhimulev, 2003), which is of interest because BLACK chromatin is particularly late-replicating. EFF is highly similar to the yeast and mammalian ubiquitin ligase Ubc4 that mediates ubiquitination of histone H3, raising the possibility that nucleosomes in BLACK chromatin may carry specific ubiquitin marks. These insights suggest that BLACK chromatin is important for chromosome architecture as well as gene repression and provide important leads for further study of this previously unknown yet prevalent type of chromatin (Filion, 2010).

In RED and YELLOW chromatin most genes are active, and the overall expression levels are similar between these two chromatin types. However, RED and YELLOW chromatin differ in many respects. One of the conspicuous distinctions is the disparate levels of H3K36me3 at active transcription units. This histone mark is thought to be laid down in the course of transcription elongation and may block the activity of cryptic promoters inside the transcription unit. Why active genes in RED chromatin lack H3K36me3 remains to be elucidated (Filion, 2010).

The remarkably high protein occupancy in RED chromatin suggests that RED domains are 'hubs' of regulatory activity. This may be related to the predominantly tissue-specific expression of genes in RED chromatin, which presumably requires many regulatory proteins. It is noted that the DamID assay integrates protein binding events over nearly 24 hours, so it is likely that not all proteins bind simultaneously; some proteins may bind only during a specific stage of the cell cycle. It is highly unlikely that the high protein occupancy in RED chromatin originates from an artifact of DamID, e.g. caused by a high accessibility of RED chromatin. First, all DamID data are corrected for accessibility using parallel Dam-only measurements. Second, several proteins, such as EFF, SU(VAR)3-9 and histone H1 exhibit lower occupancies in RED than in any other chromatin type. Third, ORC also shows a specific enrichment in RED chromatin, even though it was mapped by ChIP, by another laboratory and on another detection platform. Fourth, DamID of Gal4-DBD does not show any enrichment in RED chromatin (Filion, 2010).

RED chromatin resembles DBF binding hotspots that were previously discovered in a smaller-scale study in Drosophila cells. Discrete genomic regions targeted by many DBFs have recently also been found in mouse ES cells , hence it is tempting to speculate that an equivalent of RED chromatin may also exist in mammalian cells. Housekeeping and dynamically regulated genes in budding yeast also exhibit a dichotomy in chromatin organization which may be related to the distinction between YELLOW and RED chromatin. The observations that RED chromatin is generally the earliest to replicate and strongly enriched in ORC binding, suggest that this chromatin type may be not only involved in transcriptional regulation but also in the control of DNA replication (Filion, 2010).

This analysis of DBF binding indicates that the five chromatin types together act as a guidance system to target DBFs to specific genomic regions. This system directs DBFs to certain genomic domains even though the DBF recognition motifs are more widely distributed. It is proposed that targeting specificity is at least in part achieved through interactions of DBFs with particular partner proteins that are present in some of the five chromatin types but not in others. The observation that yeast Gal4-DBD binds its motifs with nearly equal efficiency in all five chromatin types suggests that differences in compaction among the chromatin types represent overall a minor factor in the targeting of DBFs. Although additional studies will be needed to further investigate the molecular mechanisms of DBF guidance, the identification of five principal types of chromatin provides a firm basis for future dissection of the roles of chromatin organization in global gene regulation (Filion, 2010).

SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a 'nucleosomal barrier' (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both 'repressive' and 'transient' Pol II pausing (Vorobyeva, 2012).

The mechanism of ftz-f1 transcription activation has been analyzed in S2 cells. Sequential addition and removal of ecdysone allows the DHR3 and ftz-f1 genes in these cells to be activated in accordance with their expression pattern in vivo. This system is of considerable interest, since only a few Drosophila models of activated transcription are available. It also provides the possibility of studying the mechanism of pausing in the active and repressed transcription states of the same gene, whereas previous such studies have been performed with different genes (Vorobyeva, 2012).

Pol II pausing on ftz-f1 occurs at about 1.5 kb downstream of the promoter, i.e. at a much greater distance than that described for other genes (from +30 to +100 nt). Future studies will show how widespread is this mode of pausing. It is of interest in this context that a case of Pol II pausing at 800 bp downstream of the promoter was described for the β-actin gene (Vorobyeva, 2012).

The ftz-f1 activation at the molecular level is a several-stage process. At the first stage, when the ecdysone titer and DHR3 expression are high, DHR3, SAYP, TFIID, Brahma and Pol II accumulate at the promoter. Transcription is initiated, but Pol II is paused 1.5 kb downstream of the promoter; DHR3, SAYP and Brahma are also present at this site, where a nucleosomal barrier is formed. At the next stage, ~1 h after ecdysone removal, promoter-bound factors remain at the same levels, except for SAYP (its level on the promoter decreases). Pol II and associated factors disappear from the site of pausing, and the nucleosomal barrier is eliminated, but the transcription level does not increase. The following stage is characterized by rapid intensification of transcription, which reaches a maximum within several hours; the level of Pol II increases in the body of the gene, and its pausing is observed again, with SAYP and Brahma being present at the corresponding position. In addition, the level of SAYP on the promoter is recovered, indicating that it is highly regulated at different transcription stages. The DHR3 activator is present at the site of pausing, and its level does not change upon SAYP knockdown. This is evidence that DHR3 may participate in SAYP recruitment for subsequent nucleosomal barrier formation and Pol II pausing (Vorobyeva, 2012).

The region of high nucleosome density (nucleosomal barrier) is specific for the repression stage, at which the DHR3 activator induces the assembly of the Pol II preinitiation complex on the promoter and makes paused Pol II competent for transcription initiation. Nucleosomal barrier disruption by SAYP knockdown leads to the full-length transcript synthesis, indicating that the nucleosomal barrier contributes to preventing the entry of Pol II to the transcribed region. The data show that SAYP and Brahma play the crucial role in organization of the nucleosomal barrier: this barrier coincides in location with the peak of these coactivators and disappears after SAYP knockdown, which leads to elimination of Brahma from the gene. Thus, SAYP and Brahma at the stage of repressed transcription have an important role in blocking the synthesis of full-length transcripts. Although the transcription increases upon SAYP depletion and elimination of the nucleosomal barrier, its level remains low, compared with that in the permissive state. This is evidence for the existence of different mechanisms of Pol II pausing regulation, which also correlates with the fact that the depletion of NELF, an important factor of Pol II pausing, causes a 2.5-fold increase in the transcription of hsp70 or hsp26 gene in the repressed state, which, however, does not reaches the level characteristic of a fully activated gene (Vorobyeva, 2012).

The question arises as to the structure of the nucleosomal barrier. As shown previously, the human SWI/SNF complex can not only erase nucleosomes from the template but also produce a stable remodeled dimer of mononucleosome core, with this complex being also needed for converting this product back to the cores. One may suggest that the Drosophila Brahma complex operates in the same way. In the current experiments, the level of histone H3 increased ~2-fold in the region of the nucleosomal barrier, compared with its general level on the gene, which agrees with the assumption concerning the presence of a nucleosome dimer. The fact that the region of nucleosomal barrier is significantly enriched in sequences with a high nucleosome-positioning probability indicates that DNA sequences probably contribute to organization of this barrier (Vorobyeva, 2012).

Previous experiments have revealed a relationship between Pol II pausing and the nucleosomal structure of the template. It has been shown that Pol II stops at the site where the nucleosome density is restored to the average level characteristic of the gene. However, no specific nucleosome-dense regions preventing Pol II transcription have been described as yet (Vorobyeva, 2012).

The transition to the transcription-permissive state correlates with significant rearrangements in the promoter-distal region (disappearance of Brahma, SAYP, Pol II and nucleosomal barrier at the site of Pol II pausing). However, no increase in the ftz-f1 transcription level has been observed within the first 30 min after this transition. As shown in the study on estradiol (ER)-mediated gene expression, productive transcription is preceded by an unproductive cycle (~40 min) that is necessary for promoter preparation to this process. This may be the case for ftz-f1, with a certain period of time being required for rearrangements preceding its active transcription (Vorobyeva, 2012).

At the (+;-) stage, the level of SAYP on the promoter is recovered within 2-3 h after the onset of transcription, with SAYP RNAi influencing the Brahma and TFIID levels on the promoter. Pol II pausing correlating with its Ser2 modification is again observed as the transcription level increases. Although SAYP and Brahma occur again together with paused Pol II, their function appears to be different from that at the repression stage. The nucleosomal barrier is not restored, and SAYP depletion has only a slight effect on chromatin structure (Vorobyeva, 2012).

However, SAYP depletion severely disturbs transient pausing, interfering with Ser2 phosphorylation. This impairs proper transition to productive elongation and leads to a decrease in Pol II level on the body of the gene. Thus, SAYP knockdown not only affects the level of ftz-f1 activation but also shifts the timing of its expression. The slower kinetics of transcription induction together with the slight decrease in the Pol II level on the promoter upon SAYP knockdown are evidence for the retarded Pol II passage in the coding region of the gene and, hence, for disturbances in the elongation mechanisms. Similar consequences are observed for other genes regulating on pausing mechanisms (Vorobyeva, 2012).

The results of this study show that SAYP is important for proper timing of ftz-f1 transcription during Drosophila metamorphosis. The ftz-f1 gene is a major regulator of metamorphosis, that is why its precise activation in time is crucial during development. On the whole, the data provide evidence for the important role of pausing in sequential activation of genes in cascades and indicate that this mechanism may have a general role in development (Vorobyeva, 2012).

In addition, these results also support the idea that Pol II pausing may require not only NELF and DSIF but also other factors, such as nucleosome-remodeling complexes. Interestingly, the depletion of NELF proved to result in an increased nucleosome occupancy at the promoters of some genes (Vorobyeva, 2012).

In summary, this study has found that Pol II pausing is dependent on the interplay of several molecular mechanisms, including the formation of a specific chromatin structure via the action of coactivators. These results indicate that, although Pol II pausing is a genome-wide phenomenon, the specific molecular mechanism controlling paused Pol II activity on individual genes may vary significantly (Vorobyeva, 2012).

Protein Interactions

The SNR1 and BRM proteins are present in a large [> 2 x 106 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-brmK804R) disrupts many developmental processes. An optomotor-blind (omb)-GAL4 driver was used to direct expression of UAS-brmK804R 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-brmK804R 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 brmK804R mutation behaves, therefore, as a strong dominant-negative allele. Expression of brmK804R in a variety of tissues antagonizes the function of endogenous Brm protein. Expression of a GAL4-responsive brmK804R transgene (UAS-brmK804R) 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-brmK804R 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 brmK804R 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 brmK804R-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 brmK804R 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 brmK804R (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 brmK804R 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 brmK804R 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 (Armunder) 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 Armunder 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 Armunder 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)wm4h and In(1)wm4 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, taraL4, AsxXf23, ph410, Pc3, PclD5, 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 (brmK804R) 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 brmK804R over-expression, substantiating the dominant-negative nature of the brmK804R allele. Similarly, the asf11 mutation significantly enhances the rough-eye phenotype caused by overexpression of the dominant-negative brmK804R 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 (DmcycEJP), which gives rise to adults with a rough eye phenotype. Among the dominant suppressors of DmcycEJP, 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 DmcycEJP eye phenotype. Brm complex mutants suppress the DmcycEJP 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 DmcycEJP 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 DmcycEJP 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 DmcycEJP 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 DmcycEJP 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 DmcycEJP 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).

Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex

Transcription activation by RNA polymerase II (Pol II) is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, this study has shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation (Vorobyeva, 2009).

Activation of transcription by eukaryotic Pol II requires different groups of coactivators. The primary function of coactivators is to remodel and modify the chromatin template. Thus, chromatin remodelers of the Brahma (SWI/SNF-related) family play a genome-wide role in activation of Pol II-transcribed genes. One more function of coactivators is to further recruit general transcription factors (GTFs) to form the Pol II preinitiation complex. The TFIID coactivator performs this function for most of Pol II-dependent genes (Vorobyeva, 2009).

Different coactivators recruited to the promoter assist each other and interact in a highly organized gene-specific manner. However, this important regulatory step is still poorly understood. The best studied model is that of successive one-by-one recruitment of coactivators, which, in particular, is confirmed by the fact that the recruitment of chromatin-remodeling complexes is usually a prerequisite for the efficient recruitment of GTFs to the promoter. The opposite model proposes one-time recruitment of preexisting supercomplex of several coactivators, although the composition of such supercomplexes described to date appears to be either ambiguous or incomplete (Vorobyeva, 2009).

This study describes the coactivator SAYP in Drosophila (Shidlovskii, 2005). SAYP is present at numerous sites on polytene chromosomes and colocalizes with Pol II in transcriptionally active euchromatin. SAYP homologs in various metazoans have an evolutionarily conserved core containing the SAY domain, which is involved in transcription activation, and 2 PHD fingers (Shidlovskii, 2005). Recently, SAYP was found to be associated with the chromatin-remodeling Brahma complex of the PBAP subfamily (Mohrmann, 2004; Chalkley, 2008). This study shows that SAYP interacts both with Brahma and with TFIID, assembling them into a stable supercomplex named BTFly (Brahma and TFIID in one assembly). The presence of all BTFly components is crucial for its function in transcription activation. An important fact is that highly purified BTFly contains the full set of TFIID and Brahma subunits and, therefore, is an example of a stably integrated full-set coactivator complex functioning at 2 consecutive stages of transcription activation (Vorobyeva, 2009).

BTFly includes all subunits of TFIID and Brahma (PBAP subfamily), but not comparably abundant subunits of other coactivators, and is stable in the absence of a chromatin template according to biochemical evidence. Functional cooperation of SAYP, TFIID, and Brahma in development has been verified in genetic experiments. It is estimated that approximately 20% of TFIID and a few percent of Brahma are embodied into BTFly in embryonic nuclear extracts. Apparently, BTFly-mediated transcription activation is widely used in the Drosophila genome because SAYP has been found in ~150 euchromatin sites on polytene chromosomes, all containing Pol II (Vorobyeva, 2009).

Chalkley (2008) describe SAYP as a Brahma-associated protein and did not report the presence of TFIID subunits in preparations of the Brahma complex purified using antibodies against BRM and PB. A probable explanation is that these preparations contained a manifold excess of SAYP- (and TFIID)-free Brahma, the more so that the amounts of SAYP in them were barely traceable. The current study, it was shown that SAYP directly unites Brahma and TFIID, with a relatively small proportion of Brahma being incorporated into this assembly. It was shown that SAYP-associated Brahma (i.e., its form is considered in Chalkley, 2008) is unfit for stable recruitment to the promoters (Vorobyeva, 2009).

The results of current experiments with recombinant proteins suggest a structural model with the SAY domain of SAYP taken to be the linchpin of the BTFly complex. SAY directly interacts with the TAF5 subunit of TFIID and the BAP170 subunit of Brahma, assembling them into one complex. Importantly, SAY is evolutionarily conserved, suggesting a conservation of the coupling of TFIID and Brahma in other metazoans. By means of ChIP, BTFly was revealed on the promoters of SAYP-dependent genes. The presence of all components of BTFly is crucial for its recruitment and gene activation. The recruitment of SAYP, TFIID, or Brahma in the free state is impaired. SAYP-associated Brahma in the absence of TFIID is not recruited to the SAYP-dependent promoters, although no impediments are expected in this case according to the model of a sequential recruitment of remodeling complexes and TFIID. It is concluded that BTFly functions as a single entity in transcription activation (Vorobyeva, 2009).

The coupling of TFIID and Brahma by BTFly may serve to increase the efficiency of transcription activation of a definite gene. Indeed, chromatin remodeling is crucial for transcription initiation to occur, and TFIID binding is a rate-limiting step of transcription initiation in vivo (Vorobyeva, 2009).

Thus, it is considered that the direct coupling of different activities may be an important way of controlling gene expression, which is as yet poorly understood. BTFly as a probable example of a relatively simple nuclear supercomplex appears to be a useful tool for further research in this field (Vorobyeva, 2009).

SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation

The role of metazoan coactivator SAYP in nuclear receptor-driven gene activation in the ecdysone cascade of Drosophila is considered. SAYP interacts with DHR3 nuclear receptor and activates the corresponding genes by recruiting the BTFly (Brahma and TFIID) coactivator supercomplex. The knockdown of SAYP leads to a decrease in the level of DHR3-activated transcription. DHR3 and SAYP interact during development and have multiple common targets across the genome (Vorobyeva, 2011).

This study analyzed the role of transcription coactivator SAYP and the SAYP-assembled complex in the transcription activation of several genes activated by ecdysone in Drosophila. The results obtained by different methods show that SAYP interacts with the DHR3 nuclear receptor, a component of the ecdysone cascade. In particular, their direct interaction was demonstrated in the yeast two-hybrid system. In gel filtration and co-IP experiments, a significant proportion of DHR3 proved to be associated with the high-MW SAYP-containing protein complex. The association of these factors is confirmed by data on their colocalization on polytene chromosomes as well as their coexpression and cooperation during development. These data indicate that DHR3 interacts with SAYP both in embryos and in pupae (Vorobyeva, 2011).

DHR3 and SAYP are coordinately recruited onto promoters in pupae and S2 cells. SAYP knockdown has a negative effect on the level of DHR3-driven transcription, which indicates that SAYP mediates the action of DHR3 in transcription activation. Thus, SAYP operates as a classic coactivator, which is recruited by an activator and is important for full-level gene activity. This is in agreement with the previously demonstrated mechanism of SAYP action as a component of the BTFly coactivator complex (Vorobyeva, 2009). As suggested previously, BTFly possesses specific features allowing its employment as a specific and efficient molecular machine for activation of genes in development. DHR3 acts together with other components of the ecdysone pathway to establish a specific pattern of gene activity in a restricted time window. It is suggested that the DHR3-SAYP interaction may be important for such specificity of DHR3 action (Vorobyeva, 2011).

The only known target gene activated by DHR3 is ftz-f1, but the current data indicate that DHR3 together with SAYP may be important for the expression of many other genes, since both these proteins co-occupy multiple sites on the polytene chromosomes. This study has directly shown that DHR3, via the interaction with SAYP, drives the expression of several SAYP-dependent genes in cell culture. These genes have not been recognized as components of the ecdysone cascade, which is evidence that the effect of ecdysone stimulation may be much wider than expected previously (Vorobyeva, 2011).

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).

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 snr1E1 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 snr1E1 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 brmK804R mutant phenotypes. Among 14 chromosomal deficiencies that enhanced the brmK804R rough eye phenotype, this study found that 6/14 were also dominant enhancers of the snr1E1 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 (brmK804R) or an amorphic allele (brm2), can suppress the snr1E1 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 snr1E1, 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 (brmK804R) did not uncover mutations in histone-modifying families, such as acetyltransferases, deacetylases, and methyltransferases. However, the wing patterning defect associated with snr1E1 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 snr1E1 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 snr1E1 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 snr1E1. This study observed that a loss of function mutation in lid, (lid2) dominantly suppressed, whereas a loss of function mutation in Jarid2 (CG3654EY02717) enhanced the ectopic vein phenotype associated with snr1E1. 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 snr1E1 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 (CG3654EY02717) may enhance the snr1E1 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 snr1E1 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 snr1E1 ectopic vein phenotype. Enhancement of the snr1E1 phenotype by Jarid2/CG3654EY02717 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 snr1E1 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).

Histone demethylase Utx and chromatin remodeler Brm bind directly to CBP and modulate acetylation of histone H3 lysine 27

Trithorax group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. Previous studies have shown that the Drosophila acetyltransferase CREB-binding protein (CBP; Nejire) acetylates histone H3 lysine 27 (H3K27ac), thereby directly blocking its trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) in Polycomb target genes. This study shows that H3K27ac levels also depend on other TrxG proteins, including the histone H3K27-specific demethylase Utx and the chromatin-remodeling ATPase Brahma (Brm). Utx and Brm are physically associated with CBP in vivo, and Utx, Brm, and CBP colocalize genome-wide on Polycomb response elements (PREs) and on many active Polycomb target genes marked by H3K27ac. Utx and Brm bind directly to conserved zinc fingers of CBP, suggesting that their individual activities are functionally coupled in vivo. The bromodomain-containing C terminus of Brm binds to the CBP PHD finger, enhances PHD binding to histone H3, and enhances in vitro acetylation of H3K27 by recombinant CBP. brm mutations and knockdown of Utx by RNA interference (RNAi) reduce H3K27ac levels and increase H3K27me3 levels. It is proposed that direct binding of Utx and Brm to CBP and their modulation of H3K27ac play an important role in antagonizing Polycomb silencing (Tie, 2012).

Acetylation of histone H3K27, a strong predictor of active genes, has emerged as one of the central mechanisms for antagonizing/reversing Polycomb silencing since it directly prevents trimethylation of H3K27 by PRC2. Interestingly, H3K27ac is present in animals, plants, and fungi, but H3K27me3 and PRC2 homologs are present only in animals and plants, clearly indicating that H3K27ac has functions other than preventing H3K27 methylation. This study provides evidence that the Drosophila TrxG proteins Utx and Brm modulate H3K27 acetylation by CBP. Utx and Brm are physically associated with CBP in vivo and bind directly to ZF1 and the PHD finger of CBP. Genome-wide ChIP-chip analysis revealed that the chromatin binding sites of Utx, Brm, and CBP coincide on many genes and that strong peaks of all three are highly correlated with the presence of high levels of H3K27ac. Importantly, brm mutants and RNAi knockdown of Brm in vivo result in a decrease of H3K27ac and a concomitant increase of H3K27me3. Similarly, knockdown and overexpression of Utx with no change in the CBP level are sufficient to promote, respectively, a decrease and increase in the bulk H3K27ac level. This suggests that coupled H3K27 deacetylation/trimethylation and demethylation/acetylation are dynamically antagonistic. It further suggests that regulating the balance of these opposing activities is likely to play an important role in determining whether active and silent chromatin states will be maintained or switched (Tie, 2012).

This is the first report that Utx is physically associated with CBP. While functional collaboration between Utx and CBP is required to execute the sequential reactions required to switch Polycomb target genes from silent to active states, it was not obvious that this should require that they be physically associated. The fact that they are suggests that their two reactions are more efficiently coupled. It also suggests that despite the many histone and nonhistone substrates of CBP, H3K27 acetylation on Polycomb target genes to prevent their silencing is sufficiently critical to have evolved a Utx-CBP methyl-to-acetyl switching module, perhaps to counter the complementary coupling effect of the physical association of the H3K27 deacetylase RPD3 with PRC2 to create the antagonistic acetyl-to-methyl switch. Coupling of the Utx and CBP activities may increase the fidelity of maintenance of active chromatin states of Polycomb target genes by ensuring rapid reversal of H3K27ac deacetylation and methylation by RPD3 and PRC2 that may occur either adventitiously or as part of an ongoing dynamic balance between these antagonistic activities. Such coupling could also increase the efficiency of switching Polycomb target genes from a transcriptionally silent to an active state in response to developmentally programmed signals or other cellular signals, ensuring definitive establishment of the new active transcriptional state (Tie, 2012).

While Brm is well known for the chromatin remodeling activity associated with its highly conserved ATPase domain, the current findings identify another activity of Brm associated with its highly conserved BrD-containing C terminus [Brm(1417-1634)], which binds histone H3, enhances H3 binding to the CBP PHD finger, and enhances acetylation of H3K27 by CBP in vitro. The latter effect is most likely due to the simultaneous binding of Brm to H3 and the CBP PHD finger, thereby stabilizing the H3 interaction with the CBP HAT domain. However, it cannot be ruled out that it may also reflect a direct stimulatory effect of Brm on the intrinsic activity of the CBP HAT domain. In any case, this suggests that the physical association and genome-wide colocalization of CBP and Brm do not simply reflect a spatial and temporal coordination of their separate acetylation and chromatin remodeling activities but also reflect regulatory interactions. The reduced H3K27ac level in brm2 mutants and after RNAi knockdown is consistent with the observed enhancement of CBP HAT activity in vitro. However, at this time the possibility cannot be ruled out that the loss of Brm chromatin-remodeling activity in brm2 mutants may also contribute to their reduced H3K27ac level (Tie, 2012).

The physical association of Brm with CBP and with Utx reported in this study is consistent with recent reports that BRG1 can be coimmunoprecipitated with human CBP and p300 from tumor cells and with Utx/Jmjd3 in murine EL4 T cells. However, the region of human CBP reported to bind BRG1 differs from the current findings. It was reported that BRG1 interacts directly with the human CBP fragment containing ZF3, and the proline-rich region of BRG1 is required for this interaction. The current results indicate that only ZF1 and the PHD finger of Drosophila CBP bind directly to the BrD-containing C terminus of Brm. Since the proline-rich region of Drosophila Brm was not assayed due to its insolubility, the possibility cannot be ruled out that it mediates additional contact(s) with CBP (Tie, 2012).

CBP was not found in the previously purified Drosophila Brm-containing complexes, suggesting that only a portion of Brm is physically associated with CBP or that this association may be stabilized only on chromatin and/or may be regulated by other cellular signals. Consistent with this, there are some sites detected by ChIP-chip that are enriched for Brm and Utx without CBP and there are some column fractions containing Brm and Utx without CBP. It is possible that the CBP-Brm association may also serve to recruit Brm to some sites, e.g., recruitment of human Brm or BRG1 to the beta interferon (IFN-β) promoter depends on the prior presence of CBP and leads to subsequent nucleosome remodelin (Tie, 2012).

This study found that Utx, Brm, and CBP colocalize not only with H3K27ac at many regions, including promoters, transcribed regions, PREs, and other presumed cis-regulatory elements. They are also present, albeit at lower levels, on repressed genes marked by strong H3K27me3 domains (e.g., the ANT-C and BX-C). At such sites, the H3K27me3 may be protected from Utx-mediated demethylation by the binding of PC/PRC1. Alternatively, their lower levels at repressed genes may simply result in a dynamic balance of deacetylation/methylation and demethylation/acetylation that overwhelmingly favors the former. It is also possible that Utx and CBP may also be involved in Brm-dependent transcriptional repression at some sites. Recent evidence suggests that maintenance of steady-state levels of histone acetylation is highly dynamic, and the reciprocal changes in H3K27me3 and H3K27ac levels that occur upon altering Utx or E(Z) levels suggest that maintenance of histone methylation levels may also be highly dynamic. Much remains to be discovered about the factors that regulate the demethylase and acetyltransferase activities of Utx and CBP in different chromatin environments (Tie, 2012).

The bromodomains of yeast SWI2/SNF2 and human BRG1 bind specifically to H3K14ac, indicating that this binding specificity has been highly conserved during evolution. Brm(1417-1634) also binds specifically to H3K14ac, and the BrD of Brm is required for CBP binding and histone H3 binding. The BrD-containing C termini of human and plant Brm have been reported to be functionally important in vivo. Surprisingly, the BrD of Drosophila Brm has been reported to be dispensable for viability. A brm transgene containing a deletion of the central 72 residues of the BrD can rescue the late embryonic lethality of brm2 mutants, allowing them to develop into adults. Whether the reduced H3K27ac level of these brm2 mutants is also rescued has not been determined. This rescue could indicate that the Brm BrD is functionally redundant or at least not critical for achieving adequate expression of the genes responsible for the inviability of brm2 mutants. The brm2 mutation behaves genetically as a strong hypomorphic or null allele, but the sequence alteration responsible for its phenotype has not been determined and so the precise nature of its functional deficit is unknown (Tie, 2012).

In summary, this study has shown that Utx and Brm interact directly with CBP and modulate H3K27 acetylation. Utx presumably does so indirectly, at Polycomb target genes, by providing demethylated H3K27 substrate for acetylation by CBP. Their direct physical coupling could provide obvious gains in the efficiency of their two sequential reactions and their consequent H3K27ac yield. The interaction between Brm and CBP may similarly couple their activities, but this study also presents initial evidence to suggest that the binding of the Brm BrD-containing C terminus to H3 and the CBP PHD finger may also directly enhance H3K27 acetylation through its effect on H3 binding by the CBP HAT domain. It is expected that additional TrxG proteins will modulate H3K27 acetylation, including KIS and ASH1, which have recently been shown to affect H3K27me3 levels. The broad distributions of H3K27me3 over many Polycomb target genes is mirrored by similar broad distributions of H3K27ac when those genes are active, suggesting that in addition to its general genome-wide association with active genes, it may play a more specialized dual role at Polycomb target genes, where it also dynamically antagonizes the encroachment of Polycomb silencing (Tie, 2012).

Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein-protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, a highly conserved nuclear protein, Akirin, was identified as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. These results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery (Nowak, 2012).

The data establishes Akirin as a Twist-interacting protein that promotes expression from a Twist-regulated enhancer; however, the results presented in this study also indicate that Akirin does not act solely with Twist. Analysis of salivary gland polytene chromosomes demonstrated that Akirin is associated with numerous actively transcribed gene loci. Twist is not normally expressed in salivary glands, therefore this result suggests that Akirin has roles in activation of non-Twist regulated genes. Moreover, the widespread expression of Akirin throughout the entire embryo suggests that specificity of Akirin function is determined not by restriction of Akirin expression, but rather by the associated transcription factor. Indeed, potential interactions between Akirin and other transcription factors have been described: Akirin misexpression enhances phenotypes resulting from mutations in the GATA-2 homologue pannier (Pena-Rangel, 2002). Additionally, whole genome yeast 2-hybrid analysis suggests an interaction between Akirin and Charlatan, a zinc-finger transcription factor involved in development of the peripheral nervous system. Finally, recent work has identified Akirin as a promyogenic factor and target for Myostatin regulation, as well as NF-κB target gene expression in the Drosophila innate immunity pathway. Taken together, these interactions with transcription factors other than Twist, and roles in non-Twist-dependent pathways further support a model whereby Akirin functions as a general transcription cofactor. It is proposed that the regulatory mechanism involving Akirin and the Brahma chromatin remodeling complex at specific enhancers is applicable to these transcriptional regulators in these other contexts. Further experimentation is required to validate this model (Nowak, 2012).

These studies identify Akirin as a nuclear factor that genetically interacts with the BRM complex and is required for optimal expression of the Twist-dependent Dmef2 enhancer. This association between Akirin and BRM complexes is likely the mechanism whereby Akirin is linked to gene activation. The Brahma complex (BRM) promotes gene activation by remodeling the local chromatin environment allowing components of the general transcription machinery greater accessibility to the DNA. BRM complexes are tightly associated with regions of transcriptionally active chromatin, and are associated with both promoter paused (initiating) and actively elongating RNA Polymerase II complexes throughout the Drosophila genome. Although a strong physical association of BRM complexes with RNA Polymerase II has not been confirmed, loss of BRM function leads to a severe impairment in transcription by RNA polymerase II. Based on genetic and ChIP data, it is concluded that Akirin is not a core BRM subunit, but is rather an accessory protein that is capable of interacting with BRM complexes. This conclusion is based on the observation that the distribution of Akirin and BRM subunits on polytene chromosomes do not completely overlap, and because numerous biochemical analyses of BRM complex composition to date have failed to identify Akirin as a BRM subunit. Further, no specific interaction of akirin with either BAP or PBAP complex-specific subunits was observed during myogenesis. In Drosophila, both BAP and PBAP complexes have both been linked to gene activation and repression, are present in the same cells, and perform unique, yet cooperative, functions during development. Indeed, while it was possible to observe weak physical interactions between Akirin and the Brahma core subunit in vivo, these interactions were not overly robust. Moreover, it is unknown if Akirin needs to be post-translationally modified or to further associate with other factors to mediate a physical interaction with the Brahma subunit. Further, while interactions with the core Brahma subunit were tested, it remains to be determined whether Akirin may be interacting instead with other core BRM subunits. Nevertheless, the data strongly suggests a likely association as an accessory of the BRM complex. As an accessory protein, Akirin would likely confer tissue, target, and even temporal specificity on BRM complex activity by connecting BRM complexes with a particular transcription factor for promotion of gene expression (Nowak, 2012).

The data suggest that Twist target genes have different requirements for the presence of chromatin remodeling factors during gene activation and imply that the chromatin environments at these genes are varied. This also would suggest that the local chromatin environment of a particular Twist target changes over developmental time. Further experiments will be required to validate this hypothesis. As an accessory protein, Akirin optimizes Twist transcription factor activity outputs. Akirin likely accomplishes this optimization by facilitating an interaction between Twist and BRM complexes and as such, would be predicted to change the local chromatin environment to one more favorable for transcription. The exact nature of the interface between bHLH transcription factors such as Twist and chromatin remodeling complexes such as BRM has not been previously determined. The current data would suggest that Akirin would be a suitable candidate for mediating this relationship between Twist and chromatin remodeling complexes. Mammalian SWI/SNF complexes are positively associated with bHLH transcription factor activity; however, the precise role of their remodeling activity during expression of bHLH target genes remains unclear. Whether a similar linkage via Akirin is at play with mammalian Twist during development or in a cancer context remains to be tested. Nevertheless, in keeping with the proposed model of Akirin function, the data suggest a relationship between Twist and BRM during development: twiI1/+;brm2/+ double heterozygous embryos show muscle patterning defects similar to twi1/+;akirin2/+ double heterozygotes. Also, forced expression of Twist in salivary glands and subsequent analysis of colocalization on polytene chromosomes indicated that Twist and Brahma colocalized 58% of the time, a frequency similar to that observed between Twist and Akirin (Nowak, 2012).

The finding that early (i.e., 2-4 hours) occupancy of the Moira core subunit at the Dmef2 enhancer was decreased in akirin mutants would suggest that Akirin contributes to BRM complex localization. However, co-immunoprecipitation experiments would suggest that any physical interaction between these two proteins would be either highly transient, or exquisitely sensitive to the presence of interfering factors such as protein tags. Therefore, the mechanism by which Akirin would increase Moira occupancy remains unclear. The result of such a recruitment or stabilization of BRM complexes by Akirin at Twist-target loci, would presumably result in remodeling of the local environment by BRM for optimal gene expression. Further experiments, aimed at understanding the nature of the Akirin/BRM complex association are currently underway. Together, this association between Twist, Akirin and the BRM complex would provide a novel mechanism linking chromatin remodeling factors to spatiotemporal-specific gene activation by the Twist transcription factor. This work provides another venue to investigate how changes in the chromatin environment at specific targets leads to optimal gene expression and how these local changes impact the development of specific tissues (Nowak, 2012).

brahma: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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