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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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Schematic representation of the dynamics of complex association on the proximal osteocalcin promoter. The association of key factors at the osteocalcin promoter at major points during differentiation in normal cells as determined by ChIP analysis in Fig. 4, B and C, is represented schematically. The unlabeled circles represent the invariable subunits of the SWI/SNF complex. The relative positions of the complexes are indicated arbitrarily. HDAC1 is drawn in association with the BRM complex prior to induction to indicate its specific dependence on BRM association. Dissociation of HDAC1 precedes BRM complex dissociation, and binding of an ARID1B-containing complex precedes complete dissociation of ARID1A-containing complexes, indicating the existence of a transition configuration on a partially activated promoter at around day 7.
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fig5: Schematic representation of the dynamics of complex association on the proximal osteocalcin promoter. The association of key factors at the osteocalcin promoter at major points during differentiation in normal cells as determined by ChIP analysis in Fig. 4, B and C, is represented schematically. The unlabeled circles represent the invariable subunits of the SWI/SNF complex. The relative positions of the complexes are indicated arbitrarily. HDAC1 is drawn in association with the BRM complex prior to induction to indicate its specific dependence on BRM association. Dissociation of HDAC1 precedes BRM complex dissociation, and binding of an ARID1B-containing complex precedes complete dissociation of ARID1A-containing complexes, indicating the existence of a transition configuration on a partially activated promoter at around day 7.

Mentions: Runx2 was included in the QPCR array, and notably, its expression in the non-induced cells is unaffected by depletion of either ATPase (Table 1, Footnote 2). Prior to differentiation, BRM, BRG1, RUNX2, and HDAC1 can all be seen in association with the promoter in parental cells (Fig. 4B, upper panel, lanes 3-6). Association of RUNX2 is unaffected by BRM depletion in the BRM.GG5 cell line, whereas association of HDAC1 is lost in BRM.GG5 cells. Analysis of the BRG1.D16 line shows that BRG1 complexes, although present on the repressed promoter, are not linked with association of HDAC1. (The association patterns are represented schematically in Fig. 5.) These results combined with the biological phenotype indicate that the promoter is poised for expression in non-induced cells but that expression functions are overridden by the presence of BRM-containing complexes and their HDAC1 affiliate. Depletion of BRM essentially converts the association profile of the key indicators (Fig. 4B, lanes 3-6) from the pattern characteristic of the repressed promoter in parental cells (upper panel) to the pattern characteristic of the active promoter (lower panel). The promoter in the BRM.GG5 line at day 0 is almost as active as the parental promoter at day 21 of differentiation (Fig. 2).


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

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

Schematic representation of the dynamics of complex association on the proximal osteocalcin promoter. The association of key factors at the osteocalcin promoter at major points during differentiation in normal cells as determined by ChIP analysis in Fig. 4, B and C, is represented schematically. The unlabeled circles represent the invariable subunits of the SWI/SNF complex. The relative positions of the complexes are indicated arbitrarily. HDAC1 is drawn in association with the BRM complex prior to induction to indicate its specific dependence on BRM association. Dissociation of HDAC1 precedes BRM complex dissociation, and binding of an ARID1B-containing complex precedes complete dissociation of ARID1A-containing complexes, indicating the existence of a transition configuration on a partially activated promoter at around day 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Schematic representation of the dynamics of complex association on the proximal osteocalcin promoter. The association of key factors at the osteocalcin promoter at major points during differentiation in normal cells as determined by ChIP analysis in Fig. 4, B and C, is represented schematically. The unlabeled circles represent the invariable subunits of the SWI/SNF complex. The relative positions of the complexes are indicated arbitrarily. HDAC1 is drawn in association with the BRM complex prior to induction to indicate its specific dependence on BRM association. Dissociation of HDAC1 precedes BRM complex dissociation, and binding of an ARID1B-containing complex precedes complete dissociation of ARID1A-containing complexes, indicating the existence of a transition configuration on a partially activated promoter at around day 7.
Mentions: Runx2 was included in the QPCR array, and notably, its expression in the non-induced cells is unaffected by depletion of either ATPase (Table 1, Footnote 2). Prior to differentiation, BRM, BRG1, RUNX2, and HDAC1 can all be seen in association with the promoter in parental cells (Fig. 4B, upper panel, lanes 3-6). Association of RUNX2 is unaffected by BRM depletion in the BRM.GG5 cell line, whereas association of HDAC1 is lost in BRM.GG5 cells. Analysis of the BRG1.D16 line shows that BRG1 complexes, although present on the repressed promoter, are not linked with association of HDAC1. (The association patterns are represented schematically in Fig. 5.) These results combined with the biological phenotype indicate that the promoter is poised for expression in non-induced cells but that expression functions are overridden by the presence of BRM-containing complexes and their HDAC1 affiliate. Depletion of BRM essentially converts the association profile of the key indicators (Fig. 4B, lanes 3-6) from the pattern characteristic of the repressed promoter in parental cells (upper panel) to the pattern characteristic of the active promoter (lower panel). The promoter in the BRM.GG5 line at day 0 is almost as active as the parental promoter at day 21 of differentiation (Fig. 2).

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