BIOLOGICAL OVERVIEW

brahma, a member of the trithorax group (TRX-G) was initially isolated as a suppressor of Polycomb (Kennison, 1988). While Polycomb mutants fail to silence Antennapedia complex (ANTP-C) genes, trithorax mutants reverse this phenotype by failing to activate the same genes in a genetic game of "spy versus spy." brahma mutants cause developmental defects similar to other mutants which fail to express homeotic genes after embryogenesis, such as held out wings and loss of humeral bristles (Brizuela, 1994).

What is brahma's role in gene activation? To answer this question we first digress into the arcane world of the gene activation assay. There are two kinds. One uses naked DNA, not enshrouded with proteins, and the other uses chromatin, a complex of DNA and proteins in which proteins serves to restrict the access of transcription factors to their targets, the promoters of genes. Research carried out in yeast is enlightening since many of the 12 proteins found in the SWI/SNF complex of yeast have been purified and characterized. When SWI/SNF (pronounced switch-sniff) complex proteins are tested on naked DNA, the proteins act as a DNA dependent ATPase, that is they require naked DNA to harness the energy of ATP, which is required for some aspect of their function. The involvement of yeast SWI/SNF in overcoming the repressive affects of chromatin has recently been documented (Kruger, 1993). This study shows that mutating histones H3 and H4 (See Drosophila Histone H4 and Histone H3) partly relieve the requirement of the yeast SWI/SNF complex for transcription.

Brahma is homologous to yeast SWI2, a DNA-stimulated ATPase. Although both Brahma and yeast proteins have helicase domains, in no case has it been found that the helicase is functional. When they do work, helicases use the energy of ATP to unwind DNA. Despite the lack of a functional helicase domain the SWI/SNF yeast complex is still able to activate gene transcription. Although the ATPase activity is activated by naked DNA, SWI/SNF can activate gene transcription from DNA enshrouded with protein, that is SWI/SNF is able to overcome the repressive effect of chromatin to activate gene transcription (Tamkin 1995).

The second SWI2 homolog of Drosophila, ISWI, can be contrasted to Brahma, as it exists in a different gene activating complex. The ATPase activity of ISWI is activated by chromatin and not DNA; its concentration in the cell is 1000 times greater than that of Brahma, and ISWI's molecular weight is lower than that of Brahma because it lacks a C-terminal bromodomain, an important sequence domain that probably functions in protein-protein interaction. In addition, the protein complex that includes ISWI (NURF) is lower in molecular weight than the complex containing Brahma (Tsukiyama, 1995).

brahma, like yeast SWI/SNF, is part of a large protein complex. The newly identified SNR1 of Drosophila forms part of this complex. SNR1 is a homolog of yeast SNF5. Thus it is not an isolated protein that acts to activate gene transcription from chromatin but a large multi-protein complex. Both Brahma and SNR1 proteins are present in this complex as measured by their co-immunoprecipitation from Drosophila extracts (Dingwall, 1995).

It appears then, that there are two molecular machines for gene activation in Drosophila: 1) the SWI/SNF type complex, where ATPase activity depends on DNA and 2), the NURF complex, where ATPase activity relies on chromatin. Both of these protein complexes can activate transcription from chromatin, the protein enshrouded complex between DNA and protein. To date, these two complexes have yet to be fully explored. Very likely they each play a distinctive role in development, roles research efforts have yet to describe fully.

Loss of maternal brahma function blocks oogenesis; individuals homozygous for extreme brm alleles die as late embryos with no obvious pattern defects (Brizuella, 1994). Since it has not been possible to generate embryos lacking both maternal and zygotic brm function, the exact role of brm in embryonic development is not clear. Information concerning the role of brm after embryogenesis has been derived primarily from the analysis of hypomorphic brm alleles. Individuals trans-heterozygous for certain combinations of brm alleles survive to adulthood and exhibit developmental abnormalities similar to those arising from reduced expression of Antp-C and Bx-C genes, including the transformation of first legs to second legs and the fifth abdominal segment to a more anterior identity (Bruzuella, 1994). Because the effect of complete loss of brm function had not been examined, it was unclear whether brm is also involved in other processes. To clarify the role of brm in Drosophila development, mosaic analysis has been used to determine the null phenotype of brm mutations. As an alternative approach, site-directed mutagenesis was used to generate dominant-negative brm mutations and investigate the functions of evolutionarily conserved domains within the Brm protein (Elfring, 1998).

Using mosiac analysis, it has been found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. The size and frequency of experimental clones in the head and thoracic segments are significantly reduced relative to the controls. In contrast, the frequency and size of control and experimental clones in the abdomen are similar. These data indicate that brm is essential for the development of imaginal tissues but not abdominal histoblasts. It is also possible that sufficient BRM RNA or protein persists after clone induction to allow the development of the abdominal segments. Examination of the phenotype of brm clones reveals unanticipated defects in the adult peripheral nervous system. The mechanosensory bristles of brm clones in the head, thoracic, and abdominal segments are either duplicated, stunted, or fused. In many cases, the sockets are also malformed, absent, or duplicated. No homeotic transformations have been observed in experimental clones in any of the abdominal segments. How might brm control the fate or proliferation of the cells that form mechanosensory organs? The simplest explanation is that brm regulates the activity of one or more neurogenic genes. Another possibility is that brm mutation disrupts regulation of the cell cycle. This is suggested by recent studies demonstrating that the human BRG1 and HBRM proteins cooperate with retinoblastoma tumor suppressor proteins to regulate cell cycle progression (see Retinoblastoma-family protein) (Elfring, 1998).

A dominant-negative brm mutation (DNbrm) was generated by replacing a conserved lysine in the ATP-binding site of the Brm protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the high molecular weight (2 million Daltons) Brm complex. Expression of the dominant-negative Brm protein causes peripheral nervous system defects, homeotic transformations, and decreased viability. Individuals bearing one or two copies of the dominant-negative Brm are viable, but frequently exhibit partial transformations of haltere to wing, as evidenced by an increase in haltere size and the appearance of ectopic bristles on the capitellum. Approximately one third of dominant-negative Brm adults exhibit this transformation, which is presumably caused by the decreased expression of the Ultrabithorax gene. Increasing the ratio of dominant-negative Brm to wild-type Brm to 2:1 is lethal. Thus, the dominant-negative brm mutation behaves as an antimorphic allele of brm. Expression of the dominant-negative Brm protein in patterns identical to the segmentation genes hairy or engrailed has no effect on embryonic viability or segmentation. The lack of an embryonic phenotype resulting from embryonic expression of the dominant-negative Brm protein may be caused by the high maternal expression of wild-type Brm protein, which is sufficient to allow embryogenesis to proceed to near completion in the absence of zygotic brm function. Expression of the dominant-negative protein in imaginal tissues after embryogenesis leads to greatly reduced viability. Individuals reared at 20° display partial transformation of first leg to second leg, as evidenced by a reduction in the number of sex comb teeth on the first leg. This phenotype is also seen in adults trans-heterozygous for hypomorphic brm alleles and is presumably caused by decreased expression of the Sex combs reduced (Scr) gene. Adults reared at 20° also display twinning of mechanosensory bristles, a phenotype similar to that observed in clones of brm2 tissue. Expression of the dominant negative protein also has dramatic effects on the size and morphology of the wing; mutant wings are reduced in size, and the L5 and the posterior cross-vein (PCV) are usually absent. Defects in the campaniform sensilla, a class of sensory organs important for flight, are also observed with high frequency. These defects fall into four classes: missing sensilla, duplication or triplication of sensilla, transformation of sensilla into bristles, and the appearance of ectopic sensilla. Ectopic sensilla and bristles are observed most frequently on the L3 vein. Three sensilla (L3-1, L3-2, and L3-3) and no bristles are normally found on this vein. By contrast, approximately one-half of mutant wings display one or two additional sensilla on L3. Ectopic bristles are observed on this vein in approximately one-fifth of mutant wings (Elfring, 1998).

Brahma regulates the Hippo pathway activity through forming complex with Yki-Sd and regulating the transcription of Crumbs

The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, was identified that interacts with YAP. This finding led the authors to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. Brm was found to function at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, it was demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, it was also found that crb is a downstream target of Yki-Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, this study has shown that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki (Zhu, 2014).

The core signaling cascade of Hippo pathway has been extensively studied. However, the regulatory mechanism of Yki/YAP activation remains largely elusive. This study found that Brm, a component of SWI/SNF complex, interacts with Yki and regulates organ growth in Drosophila. The findings indicated that Brm is indispensable for Yki activation to drive the expression of target genes. Interestingly, it was also found that the expression of crb is regulated by Yki-Brm complex. The ChIP assay showed that Brm and Yki physically bound to the promoter region of crb, and knockdown of Yki reduced the binding of Brm to crb promoter. Taken together, this study presents a novel feedback loop between Crb and the Yki-Brm complex. Thus, the mutual regulation between apical polarity and Hippo pathway could be critical for tissue growth and homeostasis (Zhu, 2014).

Considering the transcriptional co-dependence with RNA Polymerase II and broad localization in actively transcribed regions, the Brm complex may be present on multiple promoters and is required by global gene transcription. However, the expression of only 872 genes has been observed significantly altered by Brm knockdown. This suggests that there is a selectivity of Brm-mediated transcriptional regulation. In addition to Hippo pathway, morphogens, such as Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp), also play fundamental roles in organ patterning and growth. Therefore, the readouts of Wg, Hh and Dpp pathway in wing discs with brm knockdown were examined. Interestingly, only Wg secretion was found to be altered, which argued that there was gene specificity of Brm-mediated transcription. Many other factors including the interaction of transcription factors and co-factors, histone modifications and DNA methylation have been also shown essential in this process. Therefore, how Brm regulates the specific targets transcription needs to be further investigated (Zhu, 2014).

These data demonstrate that the enhancement of Crb levels caused by yki overexpression can be suppressed by brm knockdown. However, the overexpression of Brm alone cannot alter the Crb levels. These results suggest that the regulatory function of Brm complex is indispensable for crb but relies on Yki. Together with the fact that Brm physically associate with Yki, it is argued that Brm complex is recruited to target gene promoters through Brm-Yki interaction. Accordingly, another Drosophila transcription factor Zeste has been reported that recruits the Brm complex to chromatin to initiate transcription. Beside of Brm, Moira (Mor), another subunit of the complex, has also been reported to directly interact with Yki and occupy on the promoters of Hpo target genes, which support the working model that Yki facilitates the recruitment of Brm complex onto crb promoters. Taken together, this study draws a conclusion that the specificity of Brm for regulating Hippo targets is determined by Yki recruitment. Given that both the Brm complex and Hippo pathway are conserved in mammals, these results shed a light on a conserved interaction, which might be critical for mammalian growth and tissue homeostasis (Zhu, 2014).

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 Brm^D718A mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm and coexpression of Brm^D718A 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).

Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages

The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology. Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors (INPs) is unrestrained. This study demonstrates that Drosophila SWI/SNF chromatin remodeling Brahma (Brm) complex functions cooperatively with another chromatin remodeling factor, Histone deacetylase 3 (HDAC3) to suppress the formation of ectopic type II neuroblasts. Multiple components of the Brm complex and HDAC3 physically associate with Earmuff (Erm), a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts. Consistently, the predicted Erm-binding motif is present in most of known binding loci of Brm. Furthermore, brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth. Thus, the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts (Koe, 2014).

This study reports a critical function of the Drosophila Brm remodeling complex in suppressing the formation of ectopic type II neuroblasts in larval brains. Mutants of major components of the Brm complex, including Brm and Bap55, and RNAi targeting of several Brm components formed ectopic type II neuroblasts. Therefore, the Drosophila Brm remodeling complex displays a tumor suppressor-like function in larval brains. Multiple subunits of the SWI/SNF complex are associated with various cancers. BAP47 (homologous to Snr1) is a bona fide tumor suppressor and the gene is deleted in pediatric rhabdoid tumors. Mutations in epigenetic regulators are found in approximately half of hepatocellular carcinoma and bladder cancers, and represent a significant portion of mutated genes in medulloblastoma. Drosophila Brm complex is essential for intestinal stem cell proliferation and commitment in the adult intestine. Two other chromatin remodeling factors, Iswi and Domino control germline stem cell and somatic stem cell self-renewal in the ovary (Koe, 2014).

Brm was shown to physically associate with Erm, a type II-specific transcription factor that prevents the dedifferentiation of INPs back into neuroblasts. Furthermore, Bap60 and Snr1, two other components of the Brm complex, also physically associate with Erm in a protein complex. Therefore, this study has provided the first molecular link during the regulation of type II neuroblast lineages. It is speculated that the association with Erm may provide functional specificity of the Brm remodeling complex in type II neuroblast lineages. It was also shown that brm genetically interacts with the type II-specific transcription factor erm. Ectopic neuroblast phenotype resulting from brm knockdown was dramatically enhanced by simultaneous knockdown of erm. Furthermore, brm knockdown, similar to erm^-, can be partially suppressed by loss of Notch. These functional data suggest that Erm is a co-factor of the Brm remodeling complex in type II neuroblast lineages. However, it is uncertain how the Brm-Erm protein complex functions to prevent dedifferentiation in type II neuroblast lineages (Koe, 2014).

Bioinformatic analysis has identified a 14 bp-long motif as the de novo Erm DNA-binding motif and 202 sites out of the 270 known genomic loci harboring Brm also contain the de novo Erm DNA-binding motif. As there are many genes that are potentially co-occupied by Brm and Erm, it is possible that Brm-Erm complex results in a unique configuration of the chromatin 'landscape' in INPs to prevent INP dedifferentiation into neuroblasts. Therefore, disruption of chromatin remodelers may cause widespread changes to the transcriptome, thus amplifying the effect of the single genetic mutation (Koe, 2014).

Most class I HDACs are recruited into large multi-subunit co-repressor complexes for maximal activity. HDAC1 and 2 are found in multiple co-repressor complexes, while to date HDAC3 appears to be uniquely recruited to the Silencing mediator of retinoic and thyroid receptors (SMRT)/Nuclear receptor co-repressor (N-CoR) complex. This study reports that Drosophila HDAC3 is recruited to a novel multi-subunit complex containing Brm and Erm and that this co-repressor complex prevents dedifferentiation of INPs into type II neuroblasts. The SMRT complex appears not to be important for type II neuroblasts, as knockdown of smrter that encodes a core component of the SMRT complex neither resulted in any ectopic type II neuroblasts nor enhanced the phenotype of ectopic neuroblasts by brm knockdown. This study also showed that HDAC3 dramatically enhanced the phenotype of ectopic neuroblast upon loss of brm or snr1, two core components of the Brm complex. By identifying this novel repressor complex, this study has provided a mechanistic link between transcriptional repression and histone deacetylation during the suppression of dedifferentiation. HDACs are typically recruited by oncogenic protein complexes in lymphoma and leukemia and HDAC3 inhibitors are synergistic or additive with anticancer agents for therapeutics. The finding that HDAC3 functions cooperatively with the Brm complex in suppressing suppressing dedifferentiation of INPs into neuroblasts and induces tumors in the allograph transplantation revealed an unexpected potential involvement of HDAC3 in tumor suppression in brain tissue. It will be of interest to determine whether this effect is conserved in the mammalian central nervous system and whether it occurs in tissues other than the brain (Koe, 2014).

SWI/SNF regulates half of its targets without the need of ATP-driven nucleosome remodeling by Brahma

Brahma (BRM) is the only catalytic subunit of the SWI/SNF chromatin-remodeling complex of Drosophila melanogaster. The function of SWI/SNF in transcription has long been attributed to its ability to remodel nucleosomes, which requires the ATPase activity of BRM. However, recent studies have provided evidence for a non-catalytic function of BRM in the transcriptional regulation of a few specific genes. This study used RNA-seq and ChIP-seq to identify the BRM target genes in S2 cells, and a catalytically inactive BRM mutant (K804R) was used that is unable to hydrolyze ATP to investigate the magnitude of the non-catalytic function of BRM in transcription regulation. 49% of the BRM target genes in S2 cells are regulated through mechanisms that do not require BRM to have an ATPase activity. The catalytic and non-catalytic mechanisms of SWI/SNF regulation operate on two subsets of genes that differ in promoter architecture and are linked to different biological processes. This study shows that the non-catalytic role of SWI/SNF in transcription regulation is far more prevalent than previously anticipated and that the genes that are regulated by SWI/SNF through ATPase-dependent and ATPase-independent mechanisms have specialized roles in different cellular and developmental processes (Jordan-Pla, 2018).

The most striking observation derived from the present study in S2 cells is the high number of genes that are regulated by SWI/SNF through non-catalytic mechanisms. S2 cells cannot fully recapitulate the functions of SWI/SNF in fly development and physiology, but results from previous differential gene expression studies in flies are compatible with the broad incidence of the non-catalytic function of BRM also in vivo. In a study by Zraly (2006), only a fraction of the genes that were deregulated in flies expressing a temperature-sensitive snr1 mutant were also significantly affected in flies that expressed the catalytically inactive Brm K804R mutant. A possible interpretation of these results is that a fraction of SWI/SNF-dependent genes does not require catalytically active BRM and is therefore normally expressed in the presence of Brm K804R. This observation suggests that the ATPase-independent function of BRM is widespread in vivo (Jordan-Pla, 2018).

Single-gene studies have provided clues about the nature of the mechanisms by which SWI/SNF regulates transcription without the need of ATP-driven nucleosome remodeling by BRM. For example, SWI/SNF controls the timing of ftz-f1 transcription during metamorphosis in D. melanogaster by physically obstructing transcription elongation. In the absence of ecdysone, SWI/SNF occupies a region located in the ftz-f1 gene body and acts as a chromatin barrier that pauses RNAPII elongation. The presence of BRM in gene bodies revealed by ChIP-seq study suggests that this mechanism could be common in S2 cells. On the other hand, SWI/SNF can act by recruiting transcription regulators. For example, in the control of circadian rhythms in D. melanogaster, BRM represses the expression of the circadian genes per and tim by recruiting repressors to the promoters of these genes, and this recruitment does not require BRM's ATPase activity. At the same time, BRM increases nucleosome density in these genes through ATPase-dependent chromatin remodeling to avoid excessive deposition of repressive marks. SWI/SNF is known to interact with histone deacetylases and co-repressors, including Sin3A-HDAC, NCoR and Polycomb, and also with positive regulators of transcription such as H3K27 demethylases, the trithorax-group protein Zeste, and the Mediator. Because of its extensive interaction network, SWI/SNF has the potential to act as a platform for protein-protein interactions and, in an ATPase-independent manner, recruit other chromatin regulators to activate or repress the expression of many genes. In some genes, the catalytic and non-catalytic activities of BRM may collaborate as they do in the regulation of per and tim. In many other cases, as shown by the results, the catalytic activity of BRM is dispensable (Jordan-Pla, 2018).

The function of SWI/SNF in transcription regulation has long been attributed to the ability of this complex to remodel nucleosomes, which requires ATP hydrolysis by BRM. This study has identified hundreds of genes in the genome of Drosophila melanogaster that are regulated by SWI/SNF through mechanisms that do not require BRM to have an ATPase activity. Therefore, such mechanisms must be different from conventional ATP-dependent chromatin remodeling by SWI/SNF. Moreover, the ATPase-dependent and ATPase-independent mechanisms of transcription regulation by SWI/SNF operate on different sets of genes that have different promoter configurations and are linked to different biological functions, which reveals a novel level of specialization in the mechanisms of SWI/SNF action (Jordan-Pla, 2018).

GENE STRUCTURE

Bases in 5' UTR - 55

Exons - five

Bases in 3' UTR - 297

PROTEIN STRUCTURE

Amino Acids - 1638

Structural Domains

Brahma has both a helicase domain that functions as a DNA-dependent ATPase and a bromodomain (Tamkun, 1995). The homology to Drosophila ISWI is high in the helicase domain, but ISWI, with a lower molecular weight than Brahma lacks the C-terminal bromodomain (Elfring, 1994).

The brm gene encodes a 1638 residue protein that is similar to SNF2/SWI2, a protein involved in transcriptional activation in yeast, suggesting possible models for the role of BRM in the transcriptional activation of homeotic genes. In addition, both BRM and SNF2 contain a 77 amino acid motif (the bromodomain) that is found in other Drosophila, yeast, and human regulatory proteins and may be characteristic of a new family of regulatory proteins (Tamkun, 1992).

In humans there are two SWI2 homologs, BRG1 and hbrm. BRG1 is present in a high-molecular-mass conplex of 2MD, similar in size to the yeast SWI/SNF complex. Two separate complexes contain either BRG1 or hbrm. The purified BRG1 of hbrm complexes contain 9-12 polypeptides. Like the yeast SWI/SNF complex, the complexes purified possess an ATP-dependent nucleosome disruption activity and are able to failitate transcription factor binding to mononucleosomes. Three proteins of the complex containing BRG1 have been purified, and termed BAFs (BRG1 associated factors). Two of the three, BAF170 and BAF155 are SWI3 homologs. They contain a tryptophan-repeat domain present in two or three tandem repeats in the myb family of proteins, but do not demonstrate DNA binding activity. A second conserved region is a predicted leucine-zipper region, possibly serving as a dimerization domain. BAF60 is a homolog of yeast SWP73, a novel SWI/SNF protein, and is homologous to a C.elegans protein. The subunit composition of BRG1 complex is different when purified from several different cell types (Wang, 1996a).

date revised: 15 August 2023 

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