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Components of the human SWI/SNF complex are enriched in active chromatin and are associated with the nuclear matrix.

Reyes JC, Muchardt C, Yaniv M - J. Cell Biol. (1997)

Bottom Line: We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton.Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing.Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.

View Article: PubMed Central - PubMed

Affiliation: Unité des Virus Oncogènes, UA1644 du Centre National de la Recherche Scientifique, Département des Biotechnologies, Institut Pasteur, Paris, France.

ABSTRACT
Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation. We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton. Immunofluorescence studies revealed a punctate nuclear labeling pattern that was excluded from the nucleoli and from regions of condensed chromatin. Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.

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ATP-dependent release of hBRM from the nuclei requires a hydrolyzable form of ATP. (A) HeLa nuclei were incubated with  3 mM ATP or the same concentration of AMP-PCP or ATPγS. After a 1-h incubation at 30°C, nuclei were pelleted, and proteins from  the supernatant (S) or the pellet (P) were electrophoresed and immunoblotted with α-hBRM antibody. (B) HeLa nuclei were incubated as  in A with the indicated kinase inhibitor at 0.1 mM final concentration, except in the case of cdc2 peptide that was used at 0.1 and 10 mM.
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Figure 7: ATP-dependent release of hBRM from the nuclei requires a hydrolyzable form of ATP. (A) HeLa nuclei were incubated with 3 mM ATP or the same concentration of AMP-PCP or ATPγS. After a 1-h incubation at 30°C, nuclei were pelleted, and proteins from the supernatant (S) or the pellet (P) were electrophoresed and immunoblotted with α-hBRM antibody. (B) HeLa nuclei were incubated as in A with the indicated kinase inhibitor at 0.1 mM final concentration, except in the case of cdc2 peptide that was used at 0.1 and 10 mM.

Mentions: We further investigated the mechanisms that influence the binding of SWI/SNF complex to its targets in chromatin and in the nuclear matrix (Muchardt et al., 1996). BRG1 and hBRM are phosphorylated very late in G2 phase or at the beginning of mitosis, coinciding with their exclusion from the condensed chromatin. Therefore, we tested if incubation of isolated HeLa nuclei with mitotic cell extracts in the presence of ATP would release BRG1 and hBRM from the nuclei. As a control, nuclei were also incubated with ATP in the absence of mitotic cell extract or without ATP. Surprisingly, release of BRG1 and hBRM from the nucleus depended only on the presence of ATP but not on the presence of mitotic extract (data not shown). We therefore investigated the ATP-dependent release of hBRM and BRG1 from the nucleus using immunofluorescence. HeLa cells grown on coverslips were permeabilized in isotonic buffer supplemented with 0.5% Triton X-100, washed, and then incubated for 1 h in the presence or absence of 3 mM ATP before fixation with paraformaldehyde. Immunofluorescent staining of these cells, using α-BRG1, α-hBRM, or α-lamin B antibodies, revealed that BRG1 and hBRM, but not lamin B, were released from the nuclei of the ATP-treated cells (Fig. 6). The release of hBRM after incubation of nuclei with ATP could also be followed by immunoblotting (Fig. 7), demonstrating that hBRM was not degraded. Incubation of the nuclei with nonhydrolyzable ATP analogues like AMP-PCP or ATPγS had essentially no effect on the release of hBRM from the nucleus (Fig. 7 A). In addition, the presence of ATPγS together with ATP decreased the quantity of released hBRM (Fig. 7 A).


Components of the human SWI/SNF complex are enriched in active chromatin and are associated with the nuclear matrix.

Reyes JC, Muchardt C, Yaniv M - J. Cell Biol. (1997)

ATP-dependent release of hBRM from the nuclei requires a hydrolyzable form of ATP. (A) HeLa nuclei were incubated with  3 mM ATP or the same concentration of AMP-PCP or ATPγS. After a 1-h incubation at 30°C, nuclei were pelleted, and proteins from  the supernatant (S) or the pellet (P) were electrophoresed and immunoblotted with α-hBRM antibody. (B) HeLa nuclei were incubated as  in A with the indicated kinase inhibitor at 0.1 mM final concentration, except in the case of cdc2 peptide that was used at 0.1 and 10 mM.
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Related In: Results  -  Collection

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Figure 7: ATP-dependent release of hBRM from the nuclei requires a hydrolyzable form of ATP. (A) HeLa nuclei were incubated with 3 mM ATP or the same concentration of AMP-PCP or ATPγS. After a 1-h incubation at 30°C, nuclei were pelleted, and proteins from the supernatant (S) or the pellet (P) were electrophoresed and immunoblotted with α-hBRM antibody. (B) HeLa nuclei were incubated as in A with the indicated kinase inhibitor at 0.1 mM final concentration, except in the case of cdc2 peptide that was used at 0.1 and 10 mM.
Mentions: We further investigated the mechanisms that influence the binding of SWI/SNF complex to its targets in chromatin and in the nuclear matrix (Muchardt et al., 1996). BRG1 and hBRM are phosphorylated very late in G2 phase or at the beginning of mitosis, coinciding with their exclusion from the condensed chromatin. Therefore, we tested if incubation of isolated HeLa nuclei with mitotic cell extracts in the presence of ATP would release BRG1 and hBRM from the nuclei. As a control, nuclei were also incubated with ATP in the absence of mitotic cell extract or without ATP. Surprisingly, release of BRG1 and hBRM from the nucleus depended only on the presence of ATP but not on the presence of mitotic extract (data not shown). We therefore investigated the ATP-dependent release of hBRM and BRG1 from the nucleus using immunofluorescence. HeLa cells grown on coverslips were permeabilized in isotonic buffer supplemented with 0.5% Triton X-100, washed, and then incubated for 1 h in the presence or absence of 3 mM ATP before fixation with paraformaldehyde. Immunofluorescent staining of these cells, using α-BRG1, α-hBRM, or α-lamin B antibodies, revealed that BRG1 and hBRM, but not lamin B, were released from the nuclei of the ATP-treated cells (Fig. 6). The release of hBRM after incubation of nuclei with ATP could also be followed by immunoblotting (Fig. 7), demonstrating that hBRM was not degraded. Incubation of the nuclei with nonhydrolyzable ATP analogues like AMP-PCP or ATPγS had essentially no effect on the release of hBRM from the nucleus (Fig. 7 A). In addition, the presence of ATPγS together with ATP decreased the quantity of released hBRM (Fig. 7 A).

Bottom Line: We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton.Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing.Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.

View Article: PubMed Central - PubMed

Affiliation: Unité des Virus Oncogènes, UA1644 du Centre National de la Recherche Scientifique, Département des Biotechnologies, Institut Pasteur, Paris, France.

ABSTRACT
Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation. We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton. Immunofluorescence studies revealed a punctate nuclear labeling pattern that was excluded from the nucleoli and from regions of condensed chromatin. Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.

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