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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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Regulation of osteocalcin expression in BRM- and BRG1-depleted lines. A, parental and knockdown lines were cultured in differentiation medium; total RNA was isolated at days 0, 7, 14, 21, and 28, as indicated, and analyzed by Northern blotting with sequentially applied probes for osteocalcin (OSC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Seq, sequence. B, Northern blot analysis from three independent experiments was quantified by phosphoimaging, normalized to glyceraldehyde-3-phosphate dehydrogenase signals, averaged, and plotted as arbitrary units (AU) of phosphoimaging values. Error bars indicate the average deviation from the mean.
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fig2: Regulation of osteocalcin expression in BRM- and BRG1-depleted lines. A, parental and knockdown lines were cultured in differentiation medium; total RNA was isolated at days 0, 7, 14, 21, and 28, as indicated, and analyzed by Northern blotting with sequentially applied probes for osteocalcin (OSC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Seq, sequence. B, Northern blot analysis from three independent experiments was quantified by phosphoimaging, normalized to glyceraldehyde-3-phosphate dehydrogenase signals, averaged, and plotted as arbitrary units (AU) of phosphoimaging values. Error bars indicate the average deviation from the mean.

Mentions: Osteocalcin Is Constitutively Expressed in BRM-depleted Cells—The best studied marker of late stage differentiation in osteoblasts is the mineralized matrix component osteocalcin. The osteocalcin gene (Bglap2) is a well established model for induction of tissue-specific gene expression whose activation has been shown to be dependent on SWI/SNF complex activity in a differentiating rat osteosarcoma cell line (27, 28). To probe the molecular events underlying the phenotypes of the knockdown lines, osteocalcin expression was assessed quantitatively by Northern blot analysis. Normally, osteocalcin expression is barely detectable in non-induced cells. After induction of differentiation, expression increases dramatically in parallel with mineral deposition. The Northern blot in panel A of Fig. 2 shows the typical pattern of osteocalcin (OSC) induction in parental cells as compared with expression in BRM and BRG1 knockdown lines. The BRG1-depleted cells show greatly impaired induction of osteocalcin (lanes 11-15 as compared with lanes 1-5), correlating with the severe defect in mineralization phenotype. In contrast, BRM-depleted cells show strikingly high constitutive expression of osteocalcin (lane 6 as compared with lane 1) and rapid induction to higher levels (lanes 6-10 as compared with lanes 1-5), concordant with the accelerated mineralization phenotype. Results averaged from three independent experiments are shown quantitatively in panel B.


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

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

Regulation of osteocalcin expression in BRM- and BRG1-depleted lines. A, parental and knockdown lines were cultured in differentiation medium; total RNA was isolated at days 0, 7, 14, 21, and 28, as indicated, and analyzed by Northern blotting with sequentially applied probes for osteocalcin (OSC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Seq, sequence. B, Northern blot analysis from three independent experiments was quantified by phosphoimaging, normalized to glyceraldehyde-3-phosphate dehydrogenase signals, averaged, and plotted as arbitrary units (AU) of phosphoimaging values. Error bars indicate the average deviation from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Regulation of osteocalcin expression in BRM- and BRG1-depleted lines. A, parental and knockdown lines were cultured in differentiation medium; total RNA was isolated at days 0, 7, 14, 21, and 28, as indicated, and analyzed by Northern blotting with sequentially applied probes for osteocalcin (OSC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Seq, sequence. B, Northern blot analysis from three independent experiments was quantified by phosphoimaging, normalized to glyceraldehyde-3-phosphate dehydrogenase signals, averaged, and plotted as arbitrary units (AU) of phosphoimaging values. Error bars indicate the average deviation from the mean.
Mentions: Osteocalcin Is Constitutively Expressed in BRM-depleted Cells—The best studied marker of late stage differentiation in osteoblasts is the mineralized matrix component osteocalcin. The osteocalcin gene (Bglap2) is a well established model for induction of tissue-specific gene expression whose activation has been shown to be dependent on SWI/SNF complex activity in a differentiating rat osteosarcoma cell line (27, 28). To probe the molecular events underlying the phenotypes of the knockdown lines, osteocalcin expression was assessed quantitatively by Northern blot analysis. Normally, osteocalcin expression is barely detectable in non-induced cells. After induction of differentiation, expression increases dramatically in parallel with mineral deposition. The Northern blot in panel A of Fig. 2 shows the typical pattern of osteocalcin (OSC) induction in parental cells as compared with expression in BRM and BRG1 knockdown lines. The BRG1-depleted cells show greatly impaired induction of osteocalcin (lanes 11-15 as compared with lanes 1-5), correlating with the severe defect in mineralization phenotype. In contrast, BRM-depleted cells show strikingly high constitutive expression of osteocalcin (lane 6 as compared with lane 1) and rapid induction to higher levels (lanes 6-10 as compared with lanes 1-5), concordant with the accelerated mineralization phenotype. Results averaged from three independent experiments are shown quantitatively in panel B.

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