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Antagonistic roles for BRM and BRG1 SWI/SNF complexes in differentiation.

Flowers S, Nagl NG, Beck GR, Moran E - J. Biol. Chem. (2009)

Bottom Line: However, the basis within the complex for specificity in effecting positive versus negative changes in gene expression has only begun to be elucidated.The results reveal an unexpected role for BRM-specific 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, New Jersey Medical School-University Hospital Cancer Center, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.

ABSTRACT
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.

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Real-time PCR analysis of gene expression in BRM-depleted cells. A, expression of 84 osteogenesis-associated genes was analyzed by QPCR in wild type and BRM-depleted MC3T3-E1 cells. Genes whose expression changed by more than 4-fold in BRM-depleted cells are shown in the graph above. Most changes were activating, consistent with the phenotypic evidence that BRM complexes act predominantly to repress differentiation. B, the table shows the primers used for the ChIP analysis in panel C. C, the promoters of a selection of the genes identified in panel A were subjected to ChIP analysis with the indicated primers to determine whether the genes are direct targets of BRM complexes. Neg. control, negative control. D, serial ChIP analysis indicates that BRG1 and BRM are present simultaneously on the osteocalcin promoter. The antibodies (Ab) used in ChIP 1/ChIP 2 are indicated above the lanes.
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fig3: Real-time PCR analysis of gene expression in BRM-depleted cells. A, expression of 84 osteogenesis-associated genes was analyzed by QPCR in wild type and BRM-depleted MC3T3-E1 cells. Genes whose expression changed by more than 4-fold in BRM-depleted cells are shown in the graph above. Most changes were activating, consistent with the phenotypic evidence that BRM complexes act predominantly to repress differentiation. B, the table shows the primers used for the ChIP analysis in panel C. C, the promoters of a selection of the genes identified in panel A were subjected to ChIP analysis with the indicated primers to determine whether the genes are direct targets of BRM complexes. Neg. control, negative control. D, serial ChIP analysis indicates that BRG1 and BRM are present simultaneously on the osteocalcin promoter. The antibodies (Ab) used in ChIP 1/ChIP 2 are indicated above the lanes.

Mentions: ChIP Assays—Chromatin immunoprecipitation (ChIP) assays were performed with the EZ ChIP™ system (Upstate Cell Signaling Solutions, Lake Placid, NY), according to the manufacturer's directions, modified to include preclearing of lysates with 60 μl of a 50% slurry of protein G/salmon sperm DNA for 1 h at 4 °C, and again performed overnight. Negative controls consisted of either IgG or the viral-specific monoclonal antibody 419. Primer sequences are listed in Fig. 3B. PCR conditions were 40 cycles at for 30 s at 95 °C, 30 s at 72 °C, and 30 s at 60 °C.


Antagonistic roles for BRM and BRG1 SWI/SNF complexes in differentiation.

Flowers S, Nagl NG, Beck GR, Moran E - J. Biol. Chem. (2009)

Real-time PCR analysis of gene expression in BRM-depleted cells. A, expression of 84 osteogenesis-associated genes was analyzed by QPCR in wild type and BRM-depleted MC3T3-E1 cells. Genes whose expression changed by more than 4-fold in BRM-depleted cells are shown in the graph above. Most changes were activating, consistent with the phenotypic evidence that BRM complexes act predominantly to repress differentiation. B, the table shows the primers used for the ChIP analysis in panel C. C, the promoters of a selection of the genes identified in panel A were subjected to ChIP analysis with the indicated primers to determine whether the genes are direct targets of BRM complexes. Neg. control, negative control. D, serial ChIP analysis indicates that BRG1 and BRM are present simultaneously on the osteocalcin promoter. The antibodies (Ab) used in ChIP 1/ChIP 2 are indicated above the lanes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665061&req=5

fig3: Real-time PCR analysis of gene expression in BRM-depleted cells. A, expression of 84 osteogenesis-associated genes was analyzed by QPCR in wild type and BRM-depleted MC3T3-E1 cells. Genes whose expression changed by more than 4-fold in BRM-depleted cells are shown in the graph above. Most changes were activating, consistent with the phenotypic evidence that BRM complexes act predominantly to repress differentiation. B, the table shows the primers used for the ChIP analysis in panel C. C, the promoters of a selection of the genes identified in panel A were subjected to ChIP analysis with the indicated primers to determine whether the genes are direct targets of BRM complexes. Neg. control, negative control. D, serial ChIP analysis indicates that BRG1 and BRM are present simultaneously on the osteocalcin promoter. The antibodies (Ab) used in ChIP 1/ChIP 2 are indicated above the lanes.
Mentions: ChIP Assays—Chromatin immunoprecipitation (ChIP) assays were performed with the EZ ChIP™ system (Upstate Cell Signaling Solutions, Lake Placid, NY), according to the manufacturer's directions, modified to include preclearing of lysates with 60 μl of a 50% slurry of protein G/salmon sperm DNA for 1 h at 4 °C, and again performed overnight. Negative controls consisted of either IgG or the viral-specific monoclonal antibody 419. Primer sequences are listed in Fig. 3B. PCR conditions were 40 cycles at for 30 s at 95 °C, 30 s at 72 °C, and 30 s at 60 °C.

Bottom Line: However, the basis within the complex for specificity in effecting positive versus negative changes in gene expression has only begun to be elucidated.The results reveal an unexpected role for BRM-specific 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, New Jersey Medical School-University Hospital Cancer Center, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus