BRG1 Complex Research Articles

Brahma safeguards canalization of cardiac mesoderm differentiation.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.

Transcriptional Activation of Matricellular Protein Spondin2 (SPON2) by BRG1 in Vascular Endothelial Cells Promotes Macrophage Chemotaxis

The matricellular protein SPON2 plays diverse roles in the development of cardiovascular diseases. SPON2 is expressed in endothelial cells, but its transcription regulation in the context of atherogenesis remains incompletely appreciated. Here we report that SPON2 expression was up-regulated by pro-atherogenic stimuli (oxLDL and TNF-α) in vascular endothelia cells. In addition, endothelial SPON2 was elevated in Apoe–/– mice fed on a Western diet compared to the control mice. Induction of SPON2 in endothelial cells by pro-atherogenic stimuli was mediated by BRG1, a chromatin remodeling protein, both in vitro and in vivo. Further analysis revealed that BRG1 interacted with the sequence-specific transcription factor Egr-1 to activate SPON2 transcription. BRG1 contributed to SPON2 trans-activation by modulating chromatin structure surrounding the SPON2 promoter. Functionally, activation of SPON2 transcription by the Egr-1/BRG1 complex provided chemoattractive cues for macrophage trafficking. SPON2 depletion abrogated the ability of BRG1 or Egr-1 to stimulate endothelial derived chemoattractive cue for macrophage migration. On the contrary, recombinant SPON2 rescued endothelial chemo-attractability in the absence of BRG1 or Egr-1. In conclusion, our data have identified a novel transcriptional cascade in endothelial cells that may potentially promote macrophage recruitment and vascular inflammation leading to atherogenesis.

An HDAC9-MALAT1-BRG1 complex mediates smooth muscle dysfunction in thoracic aortic aneurysm

Thoracic aortic aneurysm (TAA) has been associated with mutations affecting members of the TGF-β signaling pathway, or components and regulators of the vascular smooth muscle cell (VSMC) actomyosin cytoskeleton. Although both clinical groups present similar phenotypes, the existence of potential common mechanisms of pathogenesis remain obscure. Here we show that mutations affecting TGF-β signaling and VSMC cytoskeleton both lead to the formation of a ternary complex comprising the histone deacetylase HDAC9, the chromatin-remodeling enzyme BRG1, and the long noncoding RNA MALAT1. The HDAC9–MALAT1–BRG1 complex binds chromatin and represses contractile protein gene expression in association with gain of histone H3-lysine 27 trimethylation modifications. Disruption of Malat1 or Hdac9 restores contractile protein expression, improves aortic mural architecture, and inhibits experimental aneurysm growth. Thus, we highlight a shared epigenetic pathway responsible for VSMC dysfunction in both forms of TAA, with potential therapeutic implication for other known HDAC9-associated vascular diseases.

Long non-coding RNA Linc-RAM enhances myogenic differentiation by interacting with MyoD

Long non-coding RNAs (lncRNAs) are important regulators of diverse biological processes. Here we report on functional identification and characterization of a novel long intergenic non-coding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to the differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD–Baf60c–Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodelling and gene expression.

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression.

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immunoprecipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal development.

Brg1 modulates enhancer activation in mesoderm lineage commitment.

The interplay between different levels of gene regulation in modulating developmental transcriptional programs, such as histone modifications and chromatin remodeling, is not well understood. Here, we show that the chromatin remodeling factor Brg1 is required for enhancer activation in mesoderm induction. In an embryonic stem cell-based directed differentiation assay, the absence of Brg1 results in a failure of cardiomyocyte differentiation and broad deregulation of lineage-specific gene expression during mesoderm induction. We find that Brg1 co-localizes with H3K27ac at distal enhancers and is required for robust H3K27 acetylation at distal enhancers that are activated during mesoderm induction. Brg1 is also required to maintain Polycomb-mediated repression of non-mesodermal developmental regulators, suggesting cooperativity between Brg1 and Polycomb complexes. Thus, Brg1 is essential for modulating active and repressive chromatin states during mesoderm lineage commitment, in particular the activation of developmentally important enhancers. These findings demonstrate interplay between chromatin remodeling complexes and histone modifications that, together, ensure robust and broad gene regulation during crucial lineage commitment decisions.

An Epigenetic “LINK(RNA)” to Pathological Cardiac Hypertrophy

Reinduction of fetal gene expression is characteristic of the failing adult heart. Han et al. (2014) demonstrate that a novel cluster of long noncoding RNAs, Myheart, protects the heart from pathological hypertrophy, involving a negative-feedback molecular circuit with the epigenetic Brg1 complex to inhibit fetal-gene reactivation.

MyomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis.

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.

NF-κB Forms a Complex with the Chromatin Remodeler BRG1 to Regulate Schwann Cell Differentiation

In the developing peripheral nervous system, axon-derived signals stimulate Schwann cells to undergo a global genetic reprogramming involving the cessation of cellular division and the upregulation of myelin genes. How such a comprehensive change in gene transcription is regulated is poorly understood. Here we report that BRG1/SMARCA4, the central helicase of the mammalian SWI/SNF-related chromatin remodeling complex, is required for Schwann cells to differentiate and form myelin, both in vitro and in vivo, in the mouse. BRG1 was highly activated in Schwann cells at early stages of myelination, and loss of the enzyme inhibited their differentiation and completely prevented myelin formation. Furthermore, we identify NF-κB as a key transcription factor that associates with the BRG1 complex in response to neuregulin 1 type III. During myelination, BRG1 was activated through the formation of a complex with NF-κB, and both proteins bound to the promoter region of Sox10, an inducer of myelination. These findings delineate a novel mechanism whereby axonal signals promote myelination through the remodeling of chromatin structure.

Abstract SY21-01: Chromatin remodeling proteins in transcription and development

Abstract The assembly of DNA with histones into chromatin provides a mechanism to regulate gene expression in time and space. The precise architecture of various DNA regulatory elements within the hierarchy of chromatin structures controls the access and activity of transacting factors, both protein and RNA. Epigenetic marks within DNA and histones allow regulatory information to act during development and across generations. The enzymes that mediate these processes are diverse and multifunctional, capable of posttranslationally modifying DNA and histones as well as remodeling the chromatin architecture. Among these enzymes the human SWI/SNF or BRG1 remodeling complex is particularly important with clear roles in signal transduction, development and cancer. In the presentation I will describe our recent work on the detailed mechanism of action of the BRG1 complex in gene expression and during early development in the mouse. We focus on the contribution that sub-domains in the BRG1 N terminus makes if protein partners outside the normal BRG1 associated Factors (BAFs) to initiate steroid receptor mediated transcription. Studies of the complex during development highlight the roles of individual BAF proteins and their contribution to organogenesis. These findings highlight the intimate connections between chromatin remodeling and epigenetics for normal healthy development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY21-01. doi:1538-7445.AM2012-SY21-01

Positive and negative regulators of the metallothionein gene (review).

Metallothioneins (MTs) are metal-binding proteins involved in diverse processes, including metal homeostasis and detoxification, the oxidative stress response and cell proliferation. Aberrant expression and silencing of these genes are important in a number of diseases. Several positive regulators of MT genes, including metal-responsive element-binding transcription factor (MTF)-1 and upstream stimulatory factor (USF)-1, have been identified and mechanisms of induction have been well described. However, the negative regulators of MT genes remain to be elucidated. Previous studies from the group of the present review have revealed that the hematopoietic master transcription factor, PU.1, directly represses the expression levels of MT genes through its epigenetic activities, and upregulation of MT results in the potent inhibition of myeloid differentiation. The present review focuses on PU.1 and several other negative regulators of this gene, including PZ120, DNA methyltransferase 3a with Mbd3 and Brg1 complex, CCAAT enhancer binding protein α and Ku protein, and describes the suppression of the MT genes through these transcription factors.

Protein Arginine Methyltransferase 5 (PRMT5) Signaling Suppresses Protein Kinase Cδ- and p38δ-dependent Signaling and Keratinocyte Differentiation

PKCδ is a key regulator of keratinocyte differentiation that activates p38δ phosphorylation leading to increased differentiation as measured by an increased expression of the structural protein involucrin. Our previous studies suggest that p38δ exists in association with protein partners. A major goal is to identify these partners and understand their role in regulating keratinocyte differentiation. In this study we use affinity purification and mass spectrometry to identify protein arginine methyltransferase 5 (PRMT5) as part of the p38δ signaling complex. PRMT5 is an arginine methyltransferase that symmetrically dimethylates arginine residues on target proteins to alter target protein function. We show that PRMT5 knockdown is associated with increased p38δ phosphorylation, suggesting that PRMT5 impacts the p38δ signaling complex. At a functional level we show that PRMT5 inhibits the PKCδ- or 12-O-tetradecanoylphorbol-13-acetate-dependent increase in human involucrin expression, and PRMT5 dimethylates proteins in the p38δ complex. Moreover, PKCδ expression reduces the PRMT5 level, suggesting that PKCδ activates differentiation in part by reducing PRMT5 level. These studies indicate antagonism between the PKCδ and PRMT5 signaling in control of keratinocyte differentiation.

Human PIH1 associates with histone H4 to mediate the glucose-dependent enhancement of pre-rRNA synthesis

Ribosome biogenesis is critical in the growth of eukaryotic cells, in which the synthesis of precursor ribosomal RNA is the first and rate-limiting step. Here, we show that human PIH1 domain-containing protein 1 (PIH1) interacts directly with histone H4 and recruits the Brg1-SWI/SNF complex via SNF5 to human rRNA genes. This process is likely involved in PIH1-dependent DNase I-hypersensitive chromatin remodeling at the core promoter of the rRNA genes. PIH1 mediates the occupancy of not only the Brg1 complex but also the Pol I complex at the core promoter and enhances transcription initiation of rRNA genes. Additionally, the interaction between PIH1 and H4K16 expels TIP5, a component of the silencing nucleolar remodeling complex (NoRC), from the core region, suggesting that PIH1 is involved in the derepression of NoRC-silenced rRNA genes. These data indicate that PIH1 is a positive regulator of human rRNA genes and is of great importance for the recovery of human cells from nutrient starvation and the transition to glucose-induced exponential growth in vivo.

Heterocomplex Formation by Arp4 and β-Actin Involved in Integrity of the Brg1 Chromatin Remodeling Complex

Although nuclear actin and Arps (actin-related proteins) are often identified as components of multi-protein chromatin-modifying enzyme complexes, such as chromatin remodeling and histone acetyltransferase (HAT) complexes, their molecular functions still remain largely elusive. Here, we investigated the role of human Arp4 (BAF53, also known as actin-like protein 6A) in Brg1-containing chromatin remodeling complexes. Depletion of Arp4 by RNA interference impaired the integrity of these complexes and accelerated the degradation of Brg1, indicating a crucial role in their maintenance, at least in certain human cell lines. We further found that Arp4 can form a heterocomplex with β-actin. Based on structural similarities between conventional actin and Arp4, and the assumption that actin-Arp4 binding might mimic actin-actin binding, we introduced a series of mutations in Arp4 that might be expected to impair its interaction with β-actin. Some of them indeed caused reduced binding to β-actin. Interestingly, such mutant Arp4 proteins also showed reduced incorporation into Brg1 complexes, and their interaction with Myc-associated complexes as well as Tip60 HAT complexes were also impaired. Based on these findings, we propose that β-actin-Arp4 complex formation might be a crucial feature in some chromatin-modifying enzyme complexes, such as the Brg1 complex.

Abstract 1094: Transcriptional activation by pRB/p107 and coordination with SWI/SNF

Abstract The retinoblastoma susceptibility gene, Rb1, is closely linked with osteosarcoma as well as retinoblastoma. The basis of such ties between individual susceptibility genes and tumors of specific tissue origin is a central question in cancer biology. Functional analysis of pRB and the closely related family members, p107 and p130, has tended to focus on repression of proliferation, but genetic evidence from our lab and others indicates an active requirement for pRB in osteoblast differentiation that might relate more directly with osteosarcoma susceptibility. Our previous studies linked pRB with activation of the early osteoblast marker, alkaline phosphatase (the product of the Akp2 gene). Here we have investigated the functional relationships between pRB and the SWI/SNF complex in Akp2 activation. Chromatin immunoprecipitation (ChIP) assays reveal the Akp2 promoter is targeted directly by all three pRB family members. Only p130 occupies the promoter prior to induction, while pRB and p107 target Akp2 directly during activation, and concurrent with association of the global co-activator p300. Akp2 is also a direct target of SWI/SNF chromatin remodeling complexes. These ATPase-powered complexes help orchestrate the programmatic shifts in gene expression necessary for progression from a self-renewing precursor state to a state of terminal differentiation. In osteoblasts, SWI/SNF complexes that repress osteogenic gene expression in order to maintain the precursor state are characterized by inclusion of the BRM ATPase. The alternative ATPase, BRG1, characterizes complexes that activate osteogenic genes during terminal differentiation. Thus, the onset of osteoblast differentiation is marked by a switch from BRM to BRG1 complexes on the Akp2 promoter. Our analysis shows that the BRM to BRG1 switch is accomplished in a precise two-step mechanism, with dissociation of BRM-containing SWI/SNF dependent on p300, and association of BRG1-containing SWI/SNF dependent on pRB/p107. Binding of RNA polymerase-II is dependent in turn on BRG1. Thus, in contrast to their widely studied role in cell cycle repression, pRB/p107 play a directly activating role in osteogenic gene expression, specifically in recruitment of the activating SWI/SNF complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1094.

MDM2 Controls the Timely Expression of Cyclin A to Regulate the Cell Cycle

Overexpression of MDM2 has been related to oncogenesis. In this communication, we present evidence to show that MDM2 controls the cell cycle-dependent expression of cyclin A by using a pathway that ensures its timely expression. MDM2 does not inhibit cyclin D or E expression. Silencing of endogenous MDM2 expression elevates cyclin A expression. The p53-binding domain of MDM2 harbors a SWIB region homologous to a conserved domain of a chromosome remodeling factor BRG1-associated protein. The SWIB domain of MDM2 inhibits cyclin A expression in a p53- and BRG1-dependent fashion, suggesting that MDM2 interferes with p53 binding of the BRG1 complex freeing it to repress cyclin A expression. Silencing of cyclin-dependent kinase (cdk) inhibitor p16 prevents MDM2-mediated inhibition of cyclin A expression, implicating its role in the process. MDM2-mediated repression of cyclin A expression induces G(1)-S arrest, which can be rescued by ectopic expression of cyclin A. Cancer cells lacking p53, p16, or BRG1 escape MDM2-mediated repression of cyclin A expression and growth arrest. Our data propose a novel mechanism by which MDM2 controls the cell cycle in normal cells and how cancer cells may escape this important safety barrier.

Antagonistic Roles for BRM and BRG1 SWI/SNF Complexes in Differentiation

The mammalian SWI/SNF chromatin-remodeling complex is essential for the multiple changes in gene expression that occur during differentiation. However, the basis within the complex for specificity in effecting positive versus negative changes in gene expression has only begun to be elucidated. The catalytic core of the complex can be either of two closely related ATPases, BRM or BRG1, with the potential that the choice of alternative subunits is a key determinant of specificity. Short hairpin RNA-mediated depletion of the ATPases was used to explore their respective roles in the well characterized multistage process of osteoblast differentiation. The results reveal an unexpected role for BRM-specific complexes. Instead of impeding differentiation as was seen with BRG1 depletion, depletion of BRM caused accelerated progression to the differentiation phenotype. Multiple tissue-specific differentiation markers, including the tightly regulated late stage marker osteocalcin, become constitutively up-regulated in BRM-depleted cells. Chromatin immunoprecipitation analysis of the osteocalcin promoter as a model for the behavior of the complexes indicates that the promoter is a direct target of both BRM- and BRG1-containing complexes. BRG1 complexes, which are required for activation, are associated with the promoter well before induction, but the concurrent presence of BRM-specific complexes overrides their activation function. BRM-specific complexes are present only on the repressed promoter and are required for association of the co-repressor HDAC1. These findings reveal an unanticipated degree of specialization of function linked with the choice of ATPase and suggest a new paradigm for the roles of the alternative subunits during differentiation.

Activity-dependent Neuroprotective Protein Constitutes a Novel Element in the SWI/SNF Chromatin Remodeling Complex

Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein.

Heat shock transcription factor (Hsf)‐4b recruits Brg1 during the G1 phase of the cell cycle and regulates the expression of heat shock proteins

Human brahma-related gene 1(Brg1) is a subunit of the switching/sucrose non-fermenting (SWI/SNF) chromatin-remodeling complex and regulates transcription during cell growth and differentiation and has been found to be mutated in many types of human cancers. Mammalian heat shock factor 1 (Hsf1), which binds conserved sequences on the promoter of the hsp70 gene when cells are exposed to various stress stimuli, utilizes Brg1-SWI/SNF complexes and stimulates transcription in vitro at the level of initiation and elongation. In contrast to the stress-inducibility of Hsf1, in vitro transcribed/translated Hsf4b binds to the heat shock element (HSE) constitutively and loses its ability to bind HSEs following stress. The regulation of Hsf4b transcriptional activity in vivo remains unclear. Here, we present evidence that Hsf4b recruits Brg1 complexes to the promoters of heat shock proteins (HSPs) under physiological growth conditions. Furthermore, in an asynchronous cell population, the association of Hsf4b with Brg1 complexes is regulated in response to activation/inactivation of the extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling pathway. Since Brg1 is also the target of mitogen-activated protein (MAP) kinases and other protein kinases and it is hyperphosphorylated and inactivated during the G2/M phase of the cell cycle, we tested whether the association of Hsf4b with Brg1 complexes is altered during the cell cycle. The results indicate that association of Hsf4b with Brg1 complexes is undetectable during G2/M; however, an Hsf4b interaction with Brg1 complexes is evident at 1-3 h after progression of cells into G1, where chromatin structure is presumed to be more accessible to transcriptional regulatory proteins. At this time, Hsf4b exhibits increased DNA-binding activity and is detectable on promoters of multiple Hsps. To determine the unique role of Hsf4b in stimulating the expression of Hsps during the cell cycle, experiments were conducted with mouse embryo fibroblasts (MEFs) deficient in individual Hsfs. The results indicate that in the absence of Hsf1 and Hsf2, Hsf4b expression in cells leads to increased ability of Hsf4b to bind HSE during G1, leading to enhanced synthesis of inducible Hsp70.

RETRACTED: Physical and Functional Interaction of DNA Methyltransferase 3A with Mbd3 and Brg1 in Mouse Lymphosarcoma Cells

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.