brahma


EVOLUTIONARY HOMOLOGS


Table of contents

Brahma homologs and the cell cycle

At the present time, three human homologs of yeast SNF/SWI proteins have been characterized: hbrm and BRG-1, homologs of SNF2/SWI2, and hSNF5, a homolog of SNF5. During mitosis, hbrm and BRG-1 are phosphorylated and excluded from the condensed chromosomes. In this phase of the cell cycle, the level of hbrm protein is also strongly reduced, whereas the level of BRG-1 remains constant. The mitotic phosphorylation of hbrm and BRG-1 is found not to disrupt the association of these proteins with hSNF5 but correlates with a decreased affinity for the nuclear structure in early M phase. It is suggested that chromosomal exclusion of the human SNF/SWI complex at the G2-M transition could be part of the mechanism leading to transcriptional arrest during mitosis (Muchardt, 1996).

Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. Using a screen that scores for phosphorylation of proteins, 20 mitotically phosphorylated proteins have been identified in Xenopus embryos; of these, 15 have sequence similarity to other proteins. Five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A); five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. Based on the examination of this broad set of mitotic phosphoproteins, three conclusions have been drawn about how the activation of CDKs regulates cell-cycle events: (1) Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins; (2) during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation, and (3) the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression (Stukenberg, 1997).

During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited. These are two processes that might be thwarted by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm, two components of human SWI/SNF (hSWI/SNF) complexes, have recently been shown to be phosphorylated during mitosis. This suggests that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, hSWI/SNF complexes were purified at different stages of the cell cycle: hSWI/SNF was found to be inactive in cells blocked in G2-M. Mitotic hSWI/SNF contains Brg1 but not hBrm, and is phosphorylated on at least two subunits, hSWI3 and Brg1 (Sif, 1998).

To test whether phosphorylation is the only modification required to inactivate the hSWI/SNF complex, a kinase was sought, capable of phosphorylating hSWI/SNF and altering its activity. Brg1 is known to be phosphorylated in unfertilized Xenopus eggs, which are naturally blocked in metaphase. Fractionation of Xenopus egg extracts revealed that a single peak of activity, which can perfectly phosphorylate Brg1, cofractionates with ERK1. To determine whether ERK1 can phosphorylate human SWI/SNF subunits, MEK1-activated ERK1 was tested for its ability to phosphorylate the hSWI/SNF complex. Wild-type GST-ERK1 can phosphorylate SWI/SNF subunits, whereas a kinase-deficient form of GST-ERK1 cannot. Other well-characterized mitotic kinases such as cdc2-cyclinA, cdc2-cyclinB and polo-like kinase 1, are unable to phosphorylate SWI/SNF subunits. When the hSWI/SNF complex that is phosphorylated in vitro by activated GST-ERK1 was tested for its ability to remodel chromatin templates, it was found to be inactive; however, when mutant GST-ERK1 (K63M) lacking kinase activity was used, hSWI/SNF was still able to remodel assembled templates in an ATP-dependent manner. Inhibition of hSWI/SNF activity depends on the presence of wild-type GST-ERK1, which is activated by GST-MEK1. hSWI/SNF which was isolated as cells traversed mitosis, regains activity when its subunits are dephosphorylated either in vitro or in vivo. It is proposed that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis (Sif, 1998).

Active ERK1 can phosphorylate (and thus inactivate) hSWI/SNF complexes. The involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathway in mitosis is not well understood; however, recently MEK1 has been shown to be involved in the golgi fragmentation that occurs during mitosis by activating a novel ERK, one associated with the golgi membrane. ERK1 can inhibit hSWI/SNF activity by phosphorylating Brg1, hBrm, and hSWI3 in vitro. These same SWI/SNF subunits are also phosphorylated in vivo. There are thirteen potential MAPK sites in Brg1, and seven in hSWI3, so it is not clear at this time what portion of these large proteins might be phosphorylated. It is possible that ERK1, or another MAPK with similar substrate specificity, might be involved in regulating hSWI/SNF activity during mitosis; however, it is also possible that other kinases are involved. Further experiments are needed to clarify the role of the MAPK family of proteins in regulating the activity of chromatin remodeling complexes in vivo (Sif, 1998).

Immunofluorescence studies reveal a punctate nuclear labeling pattern for BRG1, hBRM and hSWI that is excluded from nucleoli and from regions of condensed chromatin. There is no significan colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fraction experiments show that both soluble and insoluble active chromatin (nuclease acessible chromatin) are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins are also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in chromatin organization properties associated with matrix attachment regions. BRG1 and hBRM are phosphorylated very late in G2 phase or at the beginning of mitosis, coinciding with their release from their nuclear targets and exclusion from the condensed chromatin (Reyes, 1997).

The retinoblastoma tumor suppressor protein (See Drosophila Retinoblastoma-family protein) binds several cellular proteins involved in cell cycle progression. RB binds specifically to the protein BRG1. BRG1 contains an RB-binding motif found in viral oncoproteins and bound to the A/B pocket and the hypophosphorylated form of RB. Coimmunoprecipitation experiments suggests BRG1 associates with the RB family in vivo. In the human carcinoma cell line SW13, BRG1 exhibits tumor suppressor activity by inducing formation of flat, growth-arrested cells. This activity depends on the ability of BRG1 to cooperate and complex with RB, as both an RB-nonbinding mutant of BRG1 and the sequestration of RB by adenovirus E1A protein abolish flat cell formation (Dunaief, 1994).

Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture. A major target of the RB protein is the S-phase-inducing transcription factor E2F1 (see Drosophila E2F). RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical interaction between RB and hbrm/BRG-1. In transfected cells RB can simultaneously contact both E2F1 and hbrm, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found within the same complex in vivo. RB and hbrm cooperate to repress E2F1 activity in transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site. However, the bromodomain seems dispensable for this activity. Taken together, these results point out an unexpected role as corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G1 arrest. The domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity (Trouche, 1997).

hBRG1 and hBRM are mammalian homologs of the SNF2/SW12 yeast transcriptional activator and of Drosophila Brahma. These proteins exist in a large multisubunit complex that likely serves to remodel chromatin: in so doing, this complex facilitates the function of specific transcription factors. The retinoblastoma protein inhibits cell cycle progression by repressing transcription of specific growth-related genes. The members of the hBRG1/hBRM family of proteins interact with the pRB family of proteins, which includes pRB, p107, and p130. Interaction between the hBRG1/hBRM family with the pRB family likely influences cellular proliferation, since both hBRG1 and hBRM (but not mutants of these proteins unable to bind to pRB family members) inhibit the formation of drug-resistant colonies when transfected into a SW13 human adenocarcinoma cell line, lacking endogenous hBRG1 or hBRM. hBRM and two isoforms of hBRG1 induce the formation of flat, growth-arrested cells in a pRB family-dependent manner when introduced into SW13 cells. This flat-cell inducing activity is severely reduced by cotransfection of the wild-type E1A protein and variably reduced by the cotransfection of mutants of E1A that lack the ability to bind to some or all members of the pRB family (Strober, 1996).

The mammalian SWI-SNF complex is an evolutionarily conserved, multi-subunit machine, involved in chromatin remodelling during transcriptional activation. Within this complex, the BRM (SNF2alpha) and BRG1 (SNF2beta) proteins are mutually exclusive subunits, which are believed to affect nucleosomal structures using the energy of ATP hydrolysis. mBRG1 protein is expressed at high levels in both embryonic and extraembryonic tissues (yolk sack and alantoid) from embryos of 7.5, 9, 12, 15 and 18 days post-coitum. In contrast, mBRM is present at very low levels at all stages of development in the embryonic tissue (20- to 30-fold less than mBRG1). mBRM expression was more elevated in the extraembryonic tissue, but still lower than mBRG1 levels. This situation changes after birth; the levels of mBRM protein surpass those of mBRG1 in some organs of the adult mouse. In order to characterize possible differences in the function of BRM and BRG1, and to gain further insight into the role of BRM-containing SWI-SNF complexes, the mouse BRM gene was inactivated by homologous recombination. BRM-/- mice develop normally, suggesting that an observed up-regulation of the BRG1 protein can functionally replace BRM in the SWI-SNF complexes of mutant cells. mBRG1 protein levels in mBRM-/- brain, liver, spleen and kidneys are higher than those of the equivalent organs in wild-type mice. Strikingly, the increase in mBRG1 levels is more pronounced in organs that contain high levels of mBRM in the wild-type animals (~5- to 6-fold increase in brain, as compared with a 2-fold increase in liver and spleen). In contrast, no changes are observed in the protein levels of other subunits of the complex such as SNF5 and BAF155. Immunoprecipitaiton experiments show that mBRG1 can replace mBRM in the fraction of SWI-SNF complexes that mBRM usually occupies. Adult mutant mice are ~15% heavier than control littermates. This may be caused by increased cell proliferation, as demonstrated by a higher mitotic index detected in mutant livers. This is supported further by the observation that mutant embryonic fibroblasts are significantly deficient in their ability to arrest in the G0/G1 phase of the cell cycle in response to cell confluency or DNA damage. These studies suggest that BRM participates in the regulation of cell proliferation in adult mice (Reyes, 1998).

The Drosophila brahma gene is strongly expressed throughout embryogenesis and in pupae, but much lower amounts are present in larvae and adult flies. This is reminiscent of the expression pattern of mBRG1. The kinetics of mBRM expression seem to be the opposite: low during embryonic development and higher in adult tissues. This may explain why no alterations in the developmental program are observed in the mBRM-/- animals, especially homeotic transformations, which have been found in Drosophila brahma mutants. These data suggest that BRG1 may have a role similar to that of brahma during development. In fact, it has been shown that no viable embryonic carcinoma F9 cells lacking both copies of mBRG1 can be obtained, suggesting that during early development, when mBRM is absent, mBRG1 is an essential gene. Several observations suggest that mBRM accumulates in slowly growing or G0-arrested cells. (1) In comparison with mBRG1, mBRM expression levels throughout development, when rapid cell division occurs, are rather low. While BRG1 expression is constitutive, zygotic expression of mBRM begins at the blastocyst stage, when the first differentiation occurs in the embryonic tissues. In addition, mBRM is not expressed in ES cells or in F9 teratocarcinoma cells, which display a very short G1 phase. mBRM expression is induced in these cells upon differentiation with retinoic acid or in embryonic bodies. (2) In adult mice, mBRM is strongly expressed in post-mitotic cell types, such as neurons. (3) Serum-deprived or contact-inhibited cells from different origins (MEFs, NIH 3T3, HeLa, mouse mammary gland epithelial cells, HC11) contain 3- to 10-fold more BRM protein than exponentially growing cells. Under these conditions, BRG1 levels either remain constant or decrease. (4) BRM has been found to be down-regulated in several transformed cell lines. All of these data suggest a differential role for BRM and BRG1 in the control of genes required for quiescence or terminal differentiation. Still other experiments suggest that both proteins share similar properties. Both hBRM and hBRG1 have been shown to interact with members of the pRb family. This interaction has been mapped to an LXCXE sequence present in the C-terminal region of both proteins. Furthermore, both hBRM and hBRG1 are able to induce growth arrest of SW13 cells, which have wild-type pRb but no detectable levels of hBRM and hBRG1. These data suggest that both hBRM and hBRG1 may cooperate in pRb-dependent regulation of gene expression, BRM being used preferentially in G0-arrested cells. It has recently been shown that hBRM cooperates in Rb-E2F-mediated repression of gene expression in transient transfection studies. Consistent with this observation, it has been found that disruption of mBRM affects the balance between proliferating and non-proliferating cells in the animal. In fact, there is an increase in the fraction of S phase cells in mBRM-/- livers. Also, confluent or UV-irradiated mBRM-/- mouse embryonic fibroblasts (MEFs) are able to partially overcome G0/G1 checkpoints. Furthermore, the increase in proliferation of mBRM-/- MEFs upon DNA damage correlates with an increase in the percentage of apoptotic cells, suggesting that cells that overcome G1 arrest undergo apoptosis. Inappropriate override of G1 arrest after DNA damage or serum deprivation also leads to apoptosis in Rb-/- MEFs or in E2F-overexpressing cells, reinforcing a connection between the pRb pathway of regulation of G1/S transition and the mBRM-containing SWI-SNF complexes. How could the mBRM-associated SWI-SNF complexes suppress proliferation? An obvious possibility, deduced from the data discussed above, is that mBRM-associated SWI-SNF complexes may assist the pRb-E2F complex in repressing genes essential for S phase entry. This is a slightly unorthodox suggestion since the SWI-SNF complex has been considered until now as a transcriptional activator. However, SWI-SNF-induced accessibility of nucleosomal DNA may promote events other than transcriptional activation, and it is likely that chromatin remodelling activities are also required for binding of transcriptional repressors such as pRb-E2F complexes. It has also been shown recently that histone deacetylation is involved in pRb-dependent repression. It is possible that the BRM-containing SWI-SNF complexes might facilitate histone deacetylation by loosening the nucleosomal structure. However, the possibility that SWI-SNF complexes may be required to activate transcription of G0- or quiescence-specific genes cannot be formally excluded. These two possibilities are not mutually exclusive (Reyes, 1998 and references).

SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling. An interaction has been identified between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required for G1/S transition. BRG1 and BAF155, mammalian homologs of yeast SWI2 and SWI3, respectively, are found in cyclin E complexes and are phosphorylated by cyclin E-associated kinase activity. Overexpression of BRG1 causes growth arrest and induction of senescence-associated beta-galactosidase activity, which can be overcome by cyclin E. These results suggest that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive state (Shanahan, 1999).

The mammalian SWI-SNF complex is a chromatin-remodelling machine involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2alpha; these two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105Rb repression of E2F transcriptional activity. In the present study, the ability of various brm/SNF2alpha deletion mutants to reverse the transformed phenotype of ras-transformed fibroblasts has been studied. Deletions within the p105Rb LXCXE binding motif or the conserved bromodomain have only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105Rb interaction domain, appears to be essential in this assay. This region is also required for cooperation of brm/SNF2alpha with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2alpha to chromatin (Bourachot, 1999).

Rb forms a repressor complex containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the cell cycle, which in turn regulates exit from G1 and from S phase, respectively (Zhang, 2000).

Mutation of the yeast SWI/SNF ATPases, SWI2/SNF2, has been shown to lead to deregulation of a subset of genes, with more genes being activated by the mutations than repressed. This finding was surprising because SWI/SNF activity had previously been associated predominantly with transcriptional activation. Recent findings indicate that the corepressor HDAC can be found in the hSWI/SNF-like Mi2b nucleosome remodeling complex. These studies provide evidence that SWI/SNF-like complexes can also participate in transcriptional repression. A recent study has also shown that the c-fos promoter can be repressed by Rb and that BRG1 is required for this repression. However, the lack of E2F sites in the c-fos promoter raises the possibility that this effect of Rb and BRG1 on the c-fos promoter may be indirect. Indeed, the c-fos promoter is sensitive to growth suppression: C33a cells are growth arrested by the combination of Rb and BRG1. Thus, inhibition of c-fos expression by Rb and BRG1 in these studies may simply reflect the growth arrest that is imposed by the combination of Rb and BRG1. The results of the current study, however, demonstrate that Rb and BRG1 cooperate to inhibit genes such as cyclin E, cyclin A, and cdc2, which are known targets of Rb-E2F and participate in cell cycle progression (Zhang, 2000 and references therein).

Recent results suggest that SWI/SNF-like nucleosome remodeling complexes and related remodeling complexes can regulate both the formation and positioning of nucleosomes along a promoter. In genes such as HO in yeast, where SWI/SNF is important for transcriptional activation, it is associated with transcriptional activators and HAT activity. In contrast, when recruited by Rb into a complex with HDAC, hSWI/SNF appears to be important for repression. Thus, it is possible that SWI/SNF facilitates the action of both HAT or HDACs on nucleosomes in vivo. Alternatively, SWI/SNF may be important for both assembling or disassembling chromatin after histones in nucleosomes are modified by HAT or HDACs. It is interesting to note that in some situations Rb is associated with transcriptional activation. For example, Rb can enhance BRG1-dependent transcriptional activation by the glucocorticoid receptor. In this situation, SWI/SNF and Rb are recruited to a promoter via a transcriptional activator associated with HAT activity (the glucocorticoid receptor) (Zhang, 2000 and references therein).

Evidence is presented that HDAC and BRG1 interact with different sites on Rb, allowing formation of an HDAC-Rb-hSWI/SNF complex. It appears that phosphorylation of Rb by cyclin D/cdk4 during G1 disrupts interaction of HDAC with Rb-hSWI/SNF, allowing expression of cyclin E and progression into S phase. These results imply that cyclin E expression is a downstream target of cyclin D/cdk4. And indeed, recent studies using a 'knock-in' of the cyclin E gene into the cyclin D1 gene locus in mice have provided evidence that cyclin E is genetically downstream of cyclin D1. In contrast to Rb-HDAC, the Rb-hSWI/SNF complex is not disrupted by cyclin D/cdk4 phosphorylation of Rb, allowing it to persist into S phase where it continues to repress the cyclin A and cdc2 genes, thereby inhibiting (or delaying) movement into M phase. After cyclin E accumulates and cyclin E/cdk2 becomes active, it appears to block Rb-hSWI/SNF activity at least in part by phosphorylation of BRG1. This allows cyclin A and cdc2 expression and progression of cells into M phase. In the absence of p16ink4a, HDAC-Rb-hSWI/SNF repression of cyclin E expression is not sufficient to completely block cyclin E/cdk2 activity (because p21waf1/cip1 and p27kip1 are sequestered away from cyclin E/cdk2 by cyclin D/cdk4). Since cyclin E/cdk2 activity is not completely blocked in the p16ink4a(-) cells, they move into S phase. However, they have difficulty exiting S phase because Rb-hSWI/SNF inhibits expression of cyclin A and cdc2. This leads to additional DNA synthesis (endoreduplication). The cells eventually manage to exit S phase and undergo mitosis, but there is no cytokinesis and they arrest with multiple nuclei. It is unclear whether Rb-hSWI/SNF also has a function in regulating a step late in mitosis that leads to formation of multiple nuclei or whether the multiple nuclei are simply the result of the additional DNA synthesis that occurs before the cells precede into mitosis (Zhang, 2000 and references therein).

A recent study has provided evidence of a role for Rb and E2F in regulation of the level of cyclin B1 and thus entry into mitosis. Cyclin A/cdk2 phosphorylates the Cdh1 subunit of anaphase promoting complex (APC), blocking activity of this ubiquitin ligase, which normally degrades cyclin B1. Rb/E2F-mediated inhibition of cyclin A expression leads to activation of APC and destruction of cyclin B1. Thus, expression of cyclin A in S phase (following cyclin E/cdk2-mediated inactivation of Rb-hSWI/SNF) would lead to cyclin A/cdk2-dependent inhibition of APC, thereby triggering accumulation of cyclin B1. In support of this model, it has been demonstrated that APC remains active following mitosis, and this activity persists through G1 and is not lost until S phase (perhaps coinciding with inhibition of Rb-hSWI/SNF and the appearance of cyclin A). Loss of Rb-hSWI/SNF also results in increased expression of cdc2. Together, the increase in cyclin B1 and cdc2 would lead to accumulation of cyclin B1/cdc2, which is required for entry into mitosis. Forcibly maintaining Rb-hSWI/SNF would then be expected to inhibit or delay movement of cells from S phase to M phase by inhibiting formation of cyclin B1/cdc2 (Zhang, 2000).

E2F transcription factors play a major role in controlling mammalian cell cycle progression. A potential tumor suppressor, prohibitin, interacts with retinoblastoma protein (Rb), regulates E2F function and this activity correlates with its growth-suppressive activity. Prohibitin recruits Brg-1/Brm to E2F-responsive promoters, and this recruitment is required for the repression of E2F-mediated transcription by prohibitin. Expression of a dominant-negative Brg-1 or Brm releases prohibitin-mediated repression of E2F and relieves prohibitin-mediated growth suppression. Although prohibitin associates with, and recruits, Brg-1 and Brm independently of Rb, prohibitin/Brg-1/Brm-mediated transcriptional repression requires Rb. A viral oncoprotein, SV40 large T antigen, can reverse prohibitin-mediated suppression of E2F-mediated gene transcription, and targets prohibitin through interruption of the association between prohibitin and Brg-1/Brm without affecting the prohibitin-E2F interaction (Wang, 2002).

Nuclear reprogramming in transplanted nuclei requires BRG1

Animal cloning by nuclear transplantation in amphibia was demonstrated almost half a century ago and raised the question of the mechanisms and genes involved in nuclear reprogramming. This study demonstrates nuclear reprogramming of permeabilized human cells using extracts from Xenopus laevis eggs and early embryos. Upregulation of pluripotency markers Oct-4 and germ cell alkaline phosphatase (GCAP) occurs in 293T cells and human primary leukocytes. Reprogrammed leukocytes had a limited life span and did not express surface antigens characteristic of pluripotent cells, indicating that reprogramming was incomplete. Reprogramming activity was detected in egg and early embryo extracts until early blastula stage. Late blastula-stage extracts were not only inactive but also inhibitory to reprogramming. Screening for factors required for reprogramming identified the chromatin remodeling ATPase BRG1. Antibody depletion of BRG1 protein or expression of dominant-negative BRG1 abolished the reprogramming ability of amphibian extracts. Conversely, overexpression of BRG1 in Xenopus animal caps extended their competence from blastula to gastrula stage to respond to basic fibroblast growth factor (bFGF) treatment with induction of the mesodermal marker Xbra. Dissection of the molecular machinery using a simplified assay system may aid in achieving complete nuclear reprogramming of somatic cells for regenerative medicine (Hansis, 2004).

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. This study shows that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, this study found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc was identified that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in approximately 3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs (Shi, 2013).

Developmental regulation of SWI2 homologs

Epigenetic regulation of gene expression through modification of chromatin organization is an important mechanism in the development of eucaryotic organisms. The developmentally regulated expressions of the mouse mBRG1 and mbrm genes, which are homologous to the yeast SWI2 gene, have been investigated. Both proteins are involved in chromatin remodeling as components of the mammalian SWI/SNF complex. The analysis was performed at a time in mouse development when the formation of a functional zygotic nucleus is closely linked to extensive chromatin modifications. Reverse transcription-polymerase chain reaction (RT-PCR) analysis in mature oocytes and through the first cleavage stages shows that both genes are highly expressed as maternal products but that they subsequently exhibit considerable differences in their levels of expression when the transition to zygotic transcription occurs. Immunodetection of the two proteins with specific antibodies parallels the RT-PCR analysis. The mBRG1 protein is present throughout preimplantation development, whereas zygotic mbrm is clearly detectable only when differentiation first occurs at the blastocyst stage. At this stage, mbrm is restricted to the inner cell mass. Cell type-specific expression of mbrm is also observed after in vitro differentiation of embryonic stem cells. These results indicate that the two murine homologs of SWI2 have substantially different roles in chromatin organization during the onset of embryonic development (LeGouy, 1998).

Chromatin remodeling complexes play crucial roles in transcription and are implicated in processes including cell proliferation, differentiation and embryonic patterning. Brg1 is the catalytic subunit of the SWI/SNF chromatin remodeling complex and shows neural-enriched expression. Although early lethality of Brg1-null mice reflects its importance in embryogenesis, this phenotype has precluded further study of specific Brg1-dependent developmental processes. A requirement of Brg1 has been identified for both Xenopus primary neurogenesis and neuronal differentiation of mammalian P19 embryonic carcinoma cells. In Xenopus, loss of Brg1 function does not affect neural induction or neural cell fate determination. However, the Sox2-positive, proliferating neural progenitor cell population is expanded, and expression of a terminally differentiated neuronal marker, N-tubulin, is diminished upon loss of Brg1 activity, suggesting that Brg1 is required for neuronal differentiation. The ability of the bHLH transcription factors Ngnr1 and NeuroD to drive neuronal differentiation was also abolished by loss of Brg1 function, indicating that Brg1 is essential for the proneural activities of Ngnr1 and NeuroD. Consistent with this, dominant-negative interference with Brg1 function in P19 cells suppresses neuronal differentiation promoted by NeuroD2, showing the requirement of Brg1 for neuronal differentiation is conserved in mammalian cells. Finally, Brg1 physically associates with both Ngnr1 and NeuroD and interference with Brg1 function blocks Neurogenin3- and NeuroD2-mediated reporter gene transactivation. Together, these results demonstrate that Brg1 (and by inference the SWI/SNF complex) is required for neuronal differentiation by mediating the transcriptional activities of proneural bHLH proteins (Seo, 2005a).

Developing myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. This study shows that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 (encoding a disintegrin and metalloproteinase with thrombospondin motif) expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly (extracellular matrix) that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer (Stankunas, 2008).

SWI/SNF developmental roles

Chromatin remodeling is an important step in promoter activation during cellular lineage commitment and differentiation. The ability of the C/EBPalpha transcription factor to direct adipocyte differentiation of uncommitted fibroblast precursors and to activate SWI/SNF-dependent myeloid-specific genes depends on a domain, C/EBPalpha transactivation element III (TE-III), that binds the SWI/SNF chromatin remodeling complex. TE-III collaborates with C/EBPalpha TBP/TFIIB interaction motifs during induction of adipogenesis and adipocyte-specific gene expression. These results indicate that C/EBPalpha acts as a lineage-instructive transcription factor through SWI/SNF-dependent modification of the chromatin structure of lineage-specific genes, followed by direct promoter activation via recruitment of the basal transcription-initiation complex, and provide a mechanism by which C/EBPalpha can mediate differentiation along multiple cellular lineages (Pedersen, 2001).

The C/EBPalpha transcription factor has the capability to execute various differentiation programs. In hematopoiesis, both eosinophil and neutrophil lineage commitment can be induced by C/EBPalpha, and adipogenesis can be initiated by C/EBPalpha in uncommitted mesenchymal precursor cells or, in collaboration with PPARgamma, in myocytes. These observations suggest that C/EBPalpha provides a fundamental function generally required for the activation of specific differentiation programs, and that the collaborating factors (PPARgamma, GATA-1, Myb, PU.1) serve to direct this function to appropriate gene loci. The requirement for the SWI/SNF interacting TE-III domain of C/EBPalpha in both adipose conversion of NIH3T3 cells and in activation of SWI/SNF-dependent myeloid-specific gene expression (in the case of mim-1, in collaboration with c-Myb) provides evidence that the capacity to recruit SWI/SNF chromatin remodeling complexes is such a function. This is further supported by the previous demonstration that providing a 'selector' molecule such as c-Myb (which by itself does not recruit SWI/SNF) with an SWI/SNF recruiting domain renders it functionally independent of C/EBP activity for myeloid-specific gene activation. Together, these observations led to a proposal that SWI/SNF recruitment is an integral part of C/EBPalpha-dependent (and C/EBPß-dependent) differentiation processes. The inability of a C/EBPß molecule lacking the CR1 SWI/SNF binding domain to replace C/EBPalpha in adipogenesis, without affecting liver gene expression, provides in vivo evidence that SWI/SNF recruitment is indeed relevant for adipogenesis (Pedersen, 2001).

Vertebrate neurons have a specialized chromatin remodeling complex, bBAF, specifically containing the actin-related protein, BAF53b, which is first expressed in postmitotic neurons at about murine embryonic day 12.5 (E12.5). BAF53b is combinatorially assembled into polymorphic complexes with ubiquitous subunits including the two ATPases BRG1 and BRM. It is speculated that bBAF complexes create neuronal-specific patterns of chromatin accessibility, thereby imparting new regulatory characteristics to ubiquitous sequence-specific transcription factors in neurons (Olave, 2002).

Murine neurons have a specific chromatin remodeling complex (bBAF) based on the neuron-specific expression of BAF53b. bBAF is assembled with either BRG1 or BRM (which are expressed in all cell types). Since BAF60a, b, and c as well as BAF250a and b are all present in neurons, it is assumed that the bBAF complex is combinatorially assembled with respect to the other subunits, as well. However, definitive demonstration of combinatorial assembly of these other subunits may be very difficult because of the cellular heterogeneity of neurons. Combinatorial assembly appears to be unique to vertebrates, because Drosophila and C. elegans have only one gene encoding each subunit including BAF53, and no evidence of combinatorial assembly of the Drosophila complex has been found. Analyses of the subunits present in bBAF suggest that these complexes are unique, distinguishing themselves from other mammalian SWI/SNF-related chromatin remodeling complexes studied to date. The exclusive presence of BAF53b but not BAF53a, substoichiometric levels of BAF155, and the presence of two new putative subunits, p160 and p180, that are different from the polybromo BAF180 present in PBAF support this notion (Olave, 2002).

These studies indicate that BAF53b protein is first expressed in neuronal precursor cells that have become postmitotic. Thus, BAF53b expression appears after the determination of the neuronal lineage, but close to the time when neurons become functionally distinct. Studies of neuron cell lineage specification in the vertebrate spinal cord have indicated that homeodomain and bHLH transcription factors play critical roles. Although some of these transcription factors are expressed before the last cell cycle division of neurons (hence before BAF53b), others, such as the motor neuron specification transcription factor Islet-1, are expressed postmitotically. The polymorphic bBAF complexes present in postmitotic vertebrate neurons have the capacity to remodel chromatin in vitro and may functionally cooperate in vivo with such postmitotic lineage specification transcription factors in accessing their regulatory targets (Olave, 2002).

Purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. The conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. These findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm (Kondo, 2004).

Precise control of cell proliferation and differentiation is critical for organogenesis. Geminin (Gem) has been proposed to link cell cycle exit and differentiation as a prodifferentiation factor and plays a role in neural cell fate acquisition. The SWI/SNF chromatin-remodeling protein Brg1 has been identified as an interacting partner of Gem. Brg1 has been implicated in cell cycle withdrawal and cellular differentiation. Surprisingly, Gem was found to antagonize Brg1 activity during neurogenesis to maintain the undifferentiated cell state. Down-regulation of Gem expression normally precedes neuronal differentiation, and gain- and loss-of-function experiments in Xenopus embryos and mouse P19 cells demonstrate that Gem is essential to prevent premature neurogenesis. Misexpression of Gem also suppresses ectopic neurogenesis driven by Ngn and NeuroD. Gem's activity to block differentiation depends upon its ability to bind Brg1 and could be mediated by Gem's inhibition of proneural basic helix-loop-helix (bHLH) Brg1 interactions required for bHLH target gene activation. The data demonstrate a novel mechanism of Gem activity, through regulation of SWI/SNF chromatin-remodeling proteins, and indicate that Gem is an essential regulator of neurogenesis that can control the timing of neural progenitor differentiation and maintain the undifferentiated cell state (Seo, 2005).

Ngn and NeuroD proteins interact directly with Brg1 and require Brg1 activity to activate target gene transcription. Since Gem interacts with Brg1, whether Gem could form a higher-order complex with Brg1 and bHLH proteins or could compete with bHLH proteins for Brg1 binding was examined. In transfection and co-IP experiments, no bHLH-Gem interactions were found, while association of Brg1 and Ngn/NeuroD was observed. Therefore, it is unlikely that Gem forms a complex together with bHLH factors and Brg1. Instead, overexpression of wild-type Gem can inhibit the association of Ngn and NeuroD with Brg1. The ability of Gem to block Ngn/NeuroD and Brg1 interaction is strongly reduced for GemDelta(BD), indicating that this activity requires an intact Brg1-binding motif. In addition, while wild-type Gem can suppress the ability of Ngn3 to activate target gene transcription, GemDelta(BD) cannot. These data suggest that Gem can suppress neuronal differentiation, at least partly, by blocking association of proneural bHLHs and Brg1, and thus preventing transcriptional activation of target genes (Seo, 2005).

Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, it has been shown that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Kr├╝ppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits (Lessard, 2007).

Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. This study has identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors that are able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal and/or myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin-remodeling complexes (Skibinski, 2014).

Brg1 is involved in cell growth arrest, apoptosis and senescence

Self-renewal, proliferation and differentiation properties of stem cells are controlled by key transcription factors. However, their activity is modulated by chromatin remodeling factors that operate at the highest hierarchical level. Studies on these factors can be especially important to dissect molecular pathways governing the biology of stem cells. SWI/SNF complexes are adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes that have been shown to be required for cell cycle control, apoptosis and cell differentiation in several biological systems. This study investigated the role of these complexes in the biology of mesenchymal stem cells (MSCs). To this end, in MSCs a forced expression of the ATPase subunit of SWI/SNF (Brg1 - also known as Smarca4) by adenoviral transduction was induced, forcing a significant cell cycle arrest of MSCs in culture. This was associated with a huge increase in apoptosis that reached a peak 3 days after transduction. In addition, signs of senescence were observed in cells having ectopic Brg1 expression. At the molecular level these phenomena were associated with activation of Rb- and p53-related pathways. Inhibition of either p53 or Rb with E1A mutated proteins suggested that both Rb and p53 are indispensable for Brg1-induced senescence, whereas only p53 seems to play a role in triggering programmed cell death. Effects were examined of forced Brg1 expression on canonical MSC differentiation in adipocytes, chondrocytes and osteocytes. Brg1 did not induce cell differentiation per se; however, this protein contributed, at least in part, to the adipocyte differentiation process. In conclusion, these results suggest that whereas some ATP-dependent chromatin remodeling factors, such as ISWI complexes, promote stem cell self-renewal and conservation of an uncommitted state, others cause an escape from 'stemness' and induction of differentiation along with senescence and cell death phenomena (Napolitano, 2007).

p63 and Brg1 control developmentally regulated higher-order chromatin remodelling at the epidermal differentiation complex locus in epidermal progenitor cells

Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. This study shows that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. It was also shown that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development (Mardaryev, 2014).


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brahma: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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