A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation.

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

HMGB1 coordinates with Brahma-related gene 1 to promote epithelial-mesenchymal transition via the PI3K/Akt/mTOR pathway in BEAS-2B cells.

High mobility group protein B1 (HMGB1), a highly conserved non-histone nuclear protein, is highly expressed in fibrotic diseases; however, the role of HMGB1 in pulmonary fibrosis has not been fully elucidated. In this study, an epithelial-mesenchymal transition (EMT) model was constructed using transforming growth factor-ß1 (TGF-ß1) to stimulate BEAS-2B cells in vitro, and HMGB1 was knocked down or overexpressed to observe its effects on cell proliferation, migration and EMT. Meanwhile, string system, immunoprecipitation and immunofluorescence analyses were applied to identify and examine the relationship between HMGB1 and its potential interacting protein Brahma-related gene 1 (BRG1), and to explore the mechanism of interaction between HMGB1 and BRG1 in EMT. The results indicate that exogenous increase in HMGB1 promotes cell proliferation and migration and facilitates EMT by enhancing the PI3K/Akt/mTOR signaling pathway, whereas silencing HMGB1 has the opposite effect. Mechanistically, HMGB1 exerts these functions by interacting with BRG1, which may enhance BRG1 function and activate the PI3K/Akt/mTOR signaling pathway, thereby promoting EMT. These results suggest that HMGB1 is important for EMT and is a potential therapeutic target for the treatment of pulmonary fibrosis.

Overexpression of Both Human Sodium Iodide Symporter (NIS) and BRG1-Bromodomain Synergistically Enhances Radioiodine Sensitivity by Stabilizing p53 through NPM1 Expression.

Improved therapeutic strategies are required to minimize side effects associated with radioiodine gene therapy to avoid unnecessary damage to normal cells and radiation-induced secondary malignancies. We previously reported that codon-optimized sodium iodide symporter (oNIS) enhances absorption of I-131 and that the brahma-associated gene 1 bromodomain (BRG1-BRD) causes inefficient DNA damage repair after high-energy X-ray therapy. To increase the therapeutic effect without applying excessive radiation, we considered the combination of oNIS and BRG1-BRD as gene therapy for the most effective radioiodine treatment. The antitumor effect of I-131 with oNIS or oNIS+BRD expression was examined by tumor xenograft models along with functional assays at the cellular level. The synergistic effect of both BRG1-BRD and oNIS gene overexpression resulted in more DNA double-strand breaks and led to reduced cell proliferation/survival rates after I-131 treatment, which was mediated by the p53/p21 pathway. We found increased p53, p21, and nucleophosmin 1 (NPM1) in oNIS- and BRD-expressing cells following I-131 treatment, even though the remaining levels of citrulline and protein arginine deiminase 4 (PAD4) were unchanged at the protein level.

Genome-wide occupancy of Arabidopsis SWI/SNF chromatin remodeler SPLAYED provides insights into its interplay with its close homolog BRAHMA and Polycomb proteins.

SPLAYED (SYD) is a SWItch/Sucrose Non-Fermentable (SWI/SNF)-type chromatin remodeler identified in Arabidopsis thaliana (Arabidopsis). It is believed to play both redundant and differential roles with its closest homolog BRAHMA (BRM) in diverse plant growth and development processes. To better understand how SYD functions, we profiled the genome-wide occupancy of SYD and its impact on the global transcriptome and trimethylation of histone H3 on lysine 27 (H3K27me3). To map the global occupancy of SYD, we generated a GFP-tagged transgenic line and used it for chromatin immunoprecipitation experiments followed by next-generation sequencing, by which more than 6000 SYD target genes were identified. Through integrating SYD occupancy and transcriptome profiles, we found that SYD preferentially targets to nucleosome-free regions of expressed genes. Further analysis revealed that SYD occupancy peaks exhibit five distinct patterns, which were also shared by BRM and BAF60, a conserved SWI/SNF complex component, indicating the common target sites of these SWI/SNF chromatin remodelers and the functional relevance of such distinct patterns. To investigate the interplay between SYD and Polycomb-group (PcG) proteins, we performed a genome-wide analysis of H3K27me3 in syd-5. We observed both increases and decreases in H3K27me3 levels at a few hundred genes in syd-5 compared to wild type. Our results imply that SYD can act antagonistically or synergistically with PcG at specific genes. Together, our SYD genome-wide occupancy data and the transcriptome and H3K27me3 profiles provide a much-needed resource for dissecting SYD's crucial roles in the regulation of plant growth and development.

The chromatin remodelling ATPase BRAHMA interacts with GATA-family transcription factor GNC to regulate flowering time in Arabidopsis.

BRAHMA (BRM) is the ATPase of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelling complex, which is indispensable for transcriptional inhibition and activation, associated with vegetative and reproductive development in Arabidopsis thaliana. Here, we show that BRM directly binds to the chromatin of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), which integrates multiple flowering signals to regulate floral transition, leading to flowering. In addition, genetic and molecular analysis showed that BRM interacts with GNC (GATA, NITRATE-INDUCIBLE, CARBON METABOLISM INVOLVED), a GATA transcription factor that represses flowering by directly repressing SOC1 expression. Furthermore, BRM is recruited by GNC to directly bind to the chromatin of SOC1. The transcript level of SOC1 is elevated in brm-3, gnc, and brm-3/gnc mutants, which is associated with increased histone H3 lysine 4 tri-methylation (H3K4Me3) but decreased DNA methylation. Taken together, our results indicate that BRM associates with GNC to regulate SOC1 expression and flowering time.

Isoleucine stimulates mTOR and SREBP-1c gene expression for milk synthesis in mammary epithelial cells through BRG1-mediated chromatin remodelling.

Several amino acids can stimulate milk synthesis in mammary epithelial cells (MEC); however, the regulatory role of isoleucine (Ile) and underlying molecular mechanism remain poorly understood. In this study, we aimed to evaluate the regulatory effects of Ile on milk protein and fat synthesis in MEC and reveal the mediation mechanism of Brahma-related gene 1 (BRG1) on this regulation. Ile dose dependently affected milk protein and fat synthesis, mechanistic target of rapamycin (mTOR) phosphorylation, sterol regulatory element binding protein 1c (SREBP-1c) expression and maturation, and BRG1 protein expression in bovine MEC. Phosphatidylinositol 3 kinase (PI3K) inhibition by LY294002 treatment blocked the stimulation of Ile on BRG1 expression. BRG1 knockdown and gene activation experiments showed that it mediated the stimulation of Ile on milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c expression and maturation in MEC. ChIP-PCR analysis detected that BRG1 bound to the promoters of mTOR and SREBP-1c, and ChIP-qPCR further detected that these bindings were increased by Ile stimulation. In addition, BRG1 positively regulated the binding of H3K27ac to these two promoters, while it negatively affected the binding of H3K27me3 to these promoters. BRG1 knockdown blocked the stimulation of Ile on these two gene expressions. The expression of BRG1 was higher in mouse mammary gland in the lactating period, compared with that in the puberty or dry period. Taken together, these experimental data reveal that Ile stimulates milk protein and fat synthesis in MEC via the PI3K-BRG1-mTOR/SREBP-1c pathway.

SWI2/SNF2 chromatin remodeling ATPases SPLAYED and BRAHMA control embryo development in rice.

KEY MESSAGE: Chromatin remodeling ATPases OsSYD and OsBRM are involved in shoot establishment, and both affect OSH gene transcription. OsSYD protein interacts with RFL, but OsBRM does not. In plants, SPLAYED (SYD) and BRAHMA (BRM) encode chromatin remodeling ATPases that use the energy derived from ATP hydrolysis to restructure nucleosomes and render certain genomic regions available to transcription factors. However, the function of SYD and BRM on rice growth and development is unknown. Here, we constructed ossyd and osbrm mutants using CRISPR/Cas9 technology and analyzed the effects of mutations on rice embryo development. We discovered that the ossyd and osbrm mutants exhibited severe defects during embryonic development, whereas endosperm development was normal. These results indicated that the development of the embryo and endosperm is independent of each other. Consequently, the ossyd- and osbrm-null mutants did not germinate due to the abnormal embryos. Furthermore, we observed the embryos of ossyd- and osbrm-null mutants, and they indeed had distinct differentiation defects in shoot establishment, acquired during embryogenesis. To verify the function of OsSYD and OsBRM in embryogenesis, we measured the transcript levels of marker genes at different stages. Compared with wild type, the expression levels of multiple OSH genes were significantly reduced in the mutants, which was consistent with the defective shoot establishment phenotypes. The interaction between SYD and RICE FLORICAULA/LFY (RFL) was revealed using a yeast two-hybrid screening system, suggesting that the interaction between the LFY homolog and chromatin remodeling ATPases is ubiquitous in plants. Collectively, our findings provide the basis for elucidating the function of OsSYD and OsBRM during embryo development in rice.

Knockdown of the chromatin remodeling ATPase gene Brahma impairs the reproductive potential of the brown planthopper, Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive pests in rice-growing regions of Asia. Extensive studies have suggested that SWI/SNF chromatin remodeling ATPase Brahma (BRM) plays multiple roles in the insect model Drosophila. Yet much less is known about the physiological properties for NlBRM. In the present study, the cloned full-length cDNA of NlBRM was 5637 bp and contained an ORF of 5292 bp encoding a 194.53 kD protein. The spatiotemporal dynamics of NlBRM was investigated by qPCR, which showed that it was abundantly expressed in the egg and ovary. Then significant downregulation of NlBRM by dsRNA injection had a relatively greater impact on female survival than male. Moreover, the number of oviposition marks of the NlBRM-RNAi females were declined by 61.11% - 73.33% compared with the controls during the subsequent 5 days after dsRNA injection. Meanwhile, the number of newly hatched BPH nymphs also decreased correspondingly by 93.56% - 100%. Phenotypic analysis revealed that none of normally banana-shaped eggs were discernable in the ovaries of NlBRM-deficient females, where mRNA expression of N. lugens vitellogenin gene was also reduced. Our results demonstrated that NlBRM played a crucial role in ovarian development and fecundity of BPH, likely by regulating the vitellogenin gene in vivo, which could be as a promising target for parental RNAi-based control of this serious rice pest.

Brahma-related gene-1 promotes tubular senescence and renal fibrosis through Wnt/ß-catenin/autophagy axis.

Although accelerated cellular senescence is closely related to the progression of chronic kidney disease (CKD) and renal fibrosis, the underlying mechanisms remain largely unknown. Here, we reported that tubular aberrant expression of Brahma-related gene 1 (BRG1), an enzymatic subunit of the SWItch/Sucrose Non-Fermentable complex, is critically involved in tubular senescence and renal fibrosis. BRG1 was significantly up-regulated in the kidneys, predominantly in tubular epithelial cells, of both CKD patients and unilateral ureteral obstruction (UUO) mice. In vivo, shRNA-mediated knockdown of BRG1 significantly ameliorated renal fibrosis, improved tubular senescence, and inhibited UUO-induced activation of Wnt/ß-catenin pathway. In mouse renal tubular epithelial cells (mTECs) and primary renal tubular cells, inhibition of BRG1 diminished transforming growth factor-ß1 (TGF-ß1)-induced cellular senescence and fibrotic responses. Correspondingly, ectopic expression of BRG1 in mTECs or normal kidneys increased p16INK4a, p19ARF, and p21 expression and senescence-associated ß-galactosidase (SA-ß-gal) activity, indicating accelerated tubular senescence. Additionally, BRG1-mediated pro-fibrotic responses were largely abolished by small interfering RNA (siRNA)-mediated p16INK4a silencing in vitro or continuous senolytic treatment with ABT-263 in vivo. Moreover, BRG1 activated the Wnt/ß-catenin pathway, which further inhibited autophagy. Pharmacologic inhibition of the Wnt/ß-catenin pathway (ICG-001) or rapamycin (RAPA)-mediated activation of autophagy effectively blocked BRG1-induced tubular senescence and fibrotic responses, while bafilomycin A1 (Baf A1)-mediated inhibition of autophagy abolished the effects of ICG-001. Further, BRG1 altered the secretome of senescent tubular cells, which promoted proliferation and activation of fibroblasts. Taken together, our results indicate that BRG1 induces tubular senescence by inhibiting autophagy via the Wnt/ß-catenin pathway, which ultimately contributes to the development of renal fibrosis.

The histone variant H2A.Z and chromatin remodeler BRAHMA act coordinately and antagonistically to regulate transcription and nucleosome dynamics in Arabidopsis.

Plants adapt to environmental changes by regulating transcription and chromatin organization. The histone H2A variant H2A.Z and the SWI2/SNF2 ATPase BRAHMA (BRM) have overlapping roles in positively and negatively regulating environmentally responsive genes in Arabidopsis, but the extent of this overlap was uncharacterized. Both factors have been associated with various changes in nucleosome positioning and stability in different contexts, but their specific roles in transcriptional regulation and chromatin organization need further characterization. We show that H2A.Z and BRM co-localize at thousands of sites, where they interact both cooperatively and antagonistically in transcriptional repression and activation of genes involved in development and responses to environmental stimuli. We identified eight classes of genes that show distinct relationships between H2A.Z and BRM with respect to their roles in transcription. These include activating and silencing transcription both redundantly and antagonistically. We found that H2A.Z contributes to a range of different nucleosome properties, while BRM stabilizes nucleosomes where it binds and destabilizes or repositions flanking nucleosomes. We also found that, at many genes regulated by both BRM and H2A.Z, both factors overlap with binding sites of the light-regulated transcription factor FAR1-Related Sequence 9 (FRS9) and that a subset of these FRS9 binding sites are dependent on H2A.Z and BRM for accessibility. Collectively, we comprehensively characterized the antagonistic and cooperative contributions of H2A.Z and BRM to transcriptional regulation, and illuminated several interrelated roles in chromatin organization. The variability observed in their individual functions implies that both BRM and H2A.Z have more context-dependent roles than previously assumed.

Inhibition of microRNA-199a-5p ameliorates oxygen-glucose deprivation/reoxygenation-induced apoptosis and oxidative stress in HT22 neurons by targeting Brg1 to activate Nrf2/HO-1 signalling.

MicroRNAs (miRNAs) have emerged as critical regulators of neuronal survival during cerebral ischaemia/reperfusion injury. Accumulating evidence has shown that miR-199a-5p plays a crucial role in regulating apoptosis and survival in various cell types. However, whether miR-199a is involved in regulating neuronal survival during cerebral ischaemia/reperfusion injury remains unknown. In this study, we aimed to explore the biological role of miR-199a-5p in regulating neuronal injury induced by oxygen-glucose deprivation/reoxygenation (OGD/R), an in vitro cellular model of cerebral ischaemia and reperfusion injury. We found that miR-199a-5p expression was significantly altered in neurons in response to OGD/R treatment. Overexpression of miR-199a-5p facilitated OGD/R-induced apoptosis and reactive oxygen species (ROS) production, whereas miR-199a-5p inhibition alleviated OGD/R-induced apoptosis and ROS production. Notably, our results identified Brahma-related gene 1 (Brg1) as a target gene of miR-199a-5p. Moreover, inhibition of miR-199a-5p promoted the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signalling via targeting Brg1. However, silencing of Brg1 markedly reversed the miR-199a-5p inhibition-mediated neuroprotective effect. Taken together, our results suggest that downregulation of miR-199a-5p protects neurons from OGD/R-induced neuronal injury through upregulating Brg1 to activate Nrf2/HO-1 signalling. The miR-199a-5p/Brg1/Nrf2/HO-1 regulation axis may play an important role in regulating neuronal survival during cerebral ischaemic/reperfusion injury in vivo.

The Regulation of CIN-like TCP Transcription Factors.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1 and 2 (TCP) family proteins are the plant-specific transcription factors extensively participating in diverse developmental processes by integrating external cues with internal signals. The roles of CINCINNATA (CIN)-like TCPs are conserved in control of the morphology and size of leaves, petal development, trichome formation and plant flowering. The tight regulation of CIN-like TCP activity at transcriptional and post-transcriptional levels are central for plant developmental plasticity in response to the ever-changing environmental conditions. In this review, we summarize recent progresses with regard to the function and regulation of CIN-like TCPs. CIN-like TCPs are regulated by abiotic and biotic cues including light, temperature and pathogens. They are also finely controlled by microRNA319 (miRNA319), chromatin remodeling complexes and auxin homeostasis. The protein degradation plays critical roles in tightly controlling the activity of CIN-like TCPs as well.

PU.1 and epigenetic signals modulate 1,25-dihydroxyvitamin D3 and C/EBPα regulation of the human cathelicidin antimicrobial peptide gene in lung epithelial cells.

LL-37, the only known human cathelicidin which is encoded by the human antimicrobial peptide (CAMP) gene, plays a critical role in protection against bacterial infection. We previously demonstrated that cathelicidin is induced by 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) in human airway epithelial cells with a resultant increase in bactericidal activity. In this study we identify key factors that co-operate with 1,25(OH) 2 D 3 in the regulation of CAMP. Our results show for the first time that PU.1, the myeloid transcription factor (which has also been identified in lung epithelial cells), co-operates with the vitamin D receptor and CCAAT/enhancer binding protein α (CEBPα) to enhance the induction of CAMP in lung epithelial cells. Our findings also indicate that enhancement of 1,25(OH) 2 D 3 regulation of CAMP by histone deacetylase inhibitors involves co-operation between acetylation and chromatin remodeling through Brahma-related gene 1 (BRG1; a component of the SWItch/sucrose nonfermentable [SWI/SNF] complex). BRG1 can be an activator or repressor depending on BRG1-associated factors. Protein arginine methyltransferase 5 (PRMT5), a methlytransferase which interacts with BRG1, represses 1,25(OH) 2 D 3 induced CAMP in part through dimethylation of H4R3. Our findings identify key mediators involved in the regulation of the CAMP gene in lung epithelial cells and suggest new approaches for therapeutic manipulation of gene expression to increase the antibacterial capability of the airway.

Control of germline stem cell differentiation by Polycomb and Trithorax group genes in the niche microenvironment.

Polycomb and Trithorax group (PcG and TrxG) genes function to regulate gene transcription by maintaining a repressive or active chromatin state, respectively. This antagonistic activity is important for body patterning during embryonic development, but whether this function module has a role in adult tissues is unclear. Here, we report that in the Drosophila ovary, disruption of the Polycomb repressive complex 1 (PRC1), specifically in the supporting escort cells, causes blockage of cystoblast differentiation and germline stem cell-like tumor formation. Tumors are caused by derepression of decapentaplegic (dpp), which prevents cystoblast differentiation. Interestingly, activation of dpp in escort cells requires the function of the TrxG gene brahma (brm), suggesting that loss of PRC1 in escort cells causes Brm-dependent dpp expression. Our study suggests a requirement for balanced activity between PcG and TrxG in an adult stem cell niche, and disruption of this balance could lead to the loss of tissue homeostasis and tumorigenesis.

Association of two BRM promoter polymorphisms and smoking status with malignant pleural mesothelioma risk and prognosis.

Brahma (BRM), of the SWI/SNF complex, has two 6 to 7 bp insertion promoter polymorphisms (BRM-741/BRM-1321) that cause epigenetic BRM suppression, and are associated with risk of multiple cancers. BRM polymorphisms were genotyped in malignant pleural mesothelioma (MPM) cases and asbestos-exposed controls. Multivariable logistic regression (risk) and Cox regression (prognosis) were performed, including stratified analyses by smoking status to investigate the effect of polymorphisms on MPM risk and prognosis. Although there was no significant association overall between BRM-741/BRM-1321 and risk in patients with MPM, a differential effect by smoking status was observed (P-interaction < .001), where homozygous variants were protective (aOR of 0.18-0.28) in ever smokers, while never smokers had increased risk when carrying homozygous variants (aOR of 2.7-4.4). While there was no association between BRM polymorphisms and OS in ever-smokers, the aHR of carrying homozygous-variants of BRM-741, BRM-1321 or both were 4.0 to 8.6 in never-smokers when compared to wild-type carriers. Mechanistically, lower mRNA expression of BRM was associated with poorer general cancer prognosis. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments (ChIP) revealed high BRM insertion variant homology to MEF2 regulatory binding sites. ChIP experimentation confirmed MEF2 binding only occurs in the presence of insertion variants. DNA-affinity purification assays revealed YWHA scaffold proteins as vital to BRM mRNA expression. Never-smokers who carry BRM homozygous variants have an increased chance of developing MPM, which results in worse prognosis. In contrast, in ever-smokers, there may be a protective effect, with no difference in overall survival. Mechanisms for the interaction between BRM and smoking require further study.

Propofol post-conditioning alleviates hepatic ischaemia reperfusion injury via BRG1-mediated Nrf2/HO-1 transcriptional activation in human and mice.

To explore the effects of propofol post-conditioning (PPC) on hepatic ischaemia/reperfusion injury (HIRI) and the potential mechanisms that might be involved in the interaction of Brahma-related gene1(BRG1) and Nuclear-related factor 2(Nrf2). Patients were randomized into PPC(n = 16) and non-PPC(NPC)( n = 21) groups. Propofol(2 mg/kg) was infused within 10 min. of the onset of liver reperfusion during liver transplantation in the PPC group. Liver function tests, as well as Brg1, Nrf2, Heme oxygenase-1(HO-1) and NADPH:quinone oxidoreductase1(NQO1) expression levels were evaluated. CMV-Brg1 mice were designed to investigate the role of Brg1 overexpression during HIRI. Brg1 and Nrf2 siRNA were used to examine the relationship between Brg1 and Nrf2/HO-1 pathways in propofol-mediated effects in a human hepatocyte(L02) hypoxia/reoxygenation(H/R) model. In patients, PPC attenuated both donor liver pathological and function injury, and reducing oxidative stress markers, compared to the NPC group, 24 hrs after surgery. PPC increased liver Brg1, Nrf2, HO-1 and NQO1 expression. In mice, PPC reduced HIRI by decreasing liver oxidative stress and activating Nrf2/HO-1 pathway, accompanied by up-regulation of BRG1 expression. BRG1 overexpression activated Nrf2/HO-1 transcription in CMV-BRG1 mice during HIRI. In vitro, PPC significantly elevated expression of Nrf2, HO-1 and NQO1, resulting in a reduction of cell DCFH-DA and 8-isoprostane levels and decreased lactate dehydrogenase levels, leading to an overall increase in cell viability. Moreover, the protective effects of propofol were partially abrogated in Nrf2-knock-down or BRG1-knock-down hepatocytes. Nrf2-knock-down drastically reduced protein expression of HO-1 and NQO1, while Brg1-knock-down decreased HO-1 expression. Propofol post-conditioning alleviates HIRI through BRG1-mediated Nrf2/HO-1 transcriptional activation.

Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis.

Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex--Brahma (Brm) and Brg1--are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes.

SWI/SNF-Mediated Lineage Determination in Mesenchymal Stem Cells Confers Resistance to Osteoporosis.

Redirecting the adipogenic potential of bone marrow-derived mesenchymal stem cells to other lineages, particularly osteoblasts, is a key goal in regenerative medicine. Controlling lineage selection through chromatin remodeling complexes such as SWI/SNF, which act coordinately to establish new patterns of gene expression, would be a desirable intervention point, but the requirement for the complex in essentially every lineage pathway has generally precluded selectivity. However, a novel approach now appears possible by targeting the subset of SWI/SNF powered by the alternative ATPase, mammalian brahma (BRM). BRM is not required for development, which has hindered understanding of its contributions, but knockdown genetics here, designed to explore the hypothesis that BRM-SWI/SNF has different regulatory roles in different mesenchymal stem cell lineages, shows that depleting BRM from mesenchymal stem cells has a dramatic effect on the balance of lineage selection between osteoblasts and adipocytes. BRM depletion enhances the proportion of cells expressing markers of osteoblast precursors at the expense of cells able to differentiate along the adipocyte lineage. This effect is evident in primary bone marrow stromal cells as well as in established cell culture models. The altered precursor balance has major physiological significance, which becomes apparent as protection against age-related osteoporosis and as reduced bone marrow adiposity in adult BRM-null mice.

IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus.

Intrauterine growth restriction (IUGR) increases the risk for neurodevelopment delay and neuroendocrine reprogramming in both humans and rats. Neuroendocrine reprogramming involves the glucocorticoid receptor (GR) gene that is epigenetically regulated in the hippocampus. Using a well-characterized rodent model, we have previously shown that IUGR increases GR exon 1.7 mRNA variant and total GR expressions in male rat pup hippocampus. Epigenetic regulation of GR transcription may involve chromatin remodeling of the GR gene. A key chromatin remodeler is Brahma-related gene-1(Brg1), a member of the ATP-dependent SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Brg1 regulates gene expression by affecting nucleosome repositioning and recruiting transcriptional components to target promoters. We hypothesized that IUGR would increase hippocampal Brg1 expression and binding to GR exon 1.7 promoter, as well as alter nucleosome positioning over GR promoters in newborn male pups. Further, we hypothesized that IUGR would lead to accumulation of specificity protein 1 (Sp1) and RNA pol II at GR exon 1.7 promoter. Indeed, we found that IUGR increased Brg1 expression and binding to GR exon 1.7 promoter. We also found that increased Brg1 binding to GR exon 1.7 promoter was associated with accumulation of Sp1 and RNA pol II carboxy terminal domain pSer-5 (a marker of active transcription). Furthermore, the transcription start site of GR exon 1.7 was located within a nucleosome-depleted region. We speculate that changes in hippocampal Brg1 expression mediate GR expression and subsequently trigger neuroendocrine reprogramming in male IUGR rats.

The extended AT-hook is a novel RNA binding motif.

The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12-15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.