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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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Association of BRG1 and hBRM with the nuclear matrix in synchronized HeLa cells. (A) HeLa Cells were synchronized by  treatment with thymidine for 40 h. After release of the block (t = 0), samples were taken at the indicated time for flow cytometry analysis. 12 h later nocodazole was added at 0.1 μg/ml final concentration. (B) Synchronized cells were taken at the indicated time and total  extracts were prepared. Equal quantities of protein were subjected to SDS-PAGE in 5% acrylamide gels and immunoblotted with α-BRG1  and α-hBRM antibodies. Phosphorylated and unphosphorylated BRG1 and hBRM are indicated. (C) Synchronized cells were taken at  the indicated time and fractionated as described in Fig. 2. Cytoplasmic (Cy), chromatin (Chr), and nuclear matrix (NM) fractions were  electrophoresed in 5% acrylamide gels and immunoblotted with α-BRG1 and α-hBRM antibodies.
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Figure 5: Association of BRG1 and hBRM with the nuclear matrix in synchronized HeLa cells. (A) HeLa Cells were synchronized by treatment with thymidine for 40 h. After release of the block (t = 0), samples were taken at the indicated time for flow cytometry analysis. 12 h later nocodazole was added at 0.1 μg/ml final concentration. (B) Synchronized cells were taken at the indicated time and total extracts were prepared. Equal quantities of protein were subjected to SDS-PAGE in 5% acrylamide gels and immunoblotted with α-BRG1 and α-hBRM antibodies. Phosphorylated and unphosphorylated BRG1 and hBRM are indicated. (C) Synchronized cells were taken at the indicated time and fractionated as described in Fig. 2. Cytoplasmic (Cy), chromatin (Chr), and nuclear matrix (NM) fractions were electrophoresed in 5% acrylamide gels and immunoblotted with α-BRG1 and α-hBRM antibodies.

Mentions: We have recently demonstrated that hBRM and BRG1 are phosphorylated in mitotic cells. The phosphorylated forms of the proteins are localized in the entire cell volume with exclusion from the condensed chromosomes (Muchardt et al., 1996). To determine more accurately the moment at which phosphorylation occurs and to pinpoint whether a correlation exists between the nature of the nuclear association of the hSWI/SNF complex and the position in the cell cycle, we have fractionated nuclei of HeLa cells synchronized at various stages of the cycle. Exponentially growing HeLa cells were blocked with thymidine (S-phase arrest) for 40 h, and then released from the block. Flow cytometry analysis revealed that after 12 h ∼95% of the cells contained a 4 N chromosomal content (Fig. 5 A). However, only ∼15% of the cells had entered mitosis, as determined by phase-contrast microscopy. At that time, nocodazole was added to the medium to block the cells in mitosis. Samples were taken at different times and total extracts were prepared. Alternatively, cells were subjected to the DNase I–high salt fractionation protocol described above. As shown in Fig. 5 B, both BRG1 and hBRM were phosphorylated at the time when cells entered mitosis but not in S and G2 phases. Both BRG1 and hBRM remained associated with the nuclear matrix during G1, S, and G2 phases (Fig. 5 C). However, all of the signal appeared in the cytoplasmic fraction in mitotic cells, consistent with the breakdown of the nuclear structure at mitosis and the exclusion of hBRM and BRG1 from the condensed chromatin as previously published (Muchardt et al., 1996).


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)

Association of BRG1 and hBRM with the nuclear matrix in synchronized HeLa cells. (A) HeLa Cells were synchronized by  treatment with thymidine for 40 h. After release of the block (t = 0), samples were taken at the indicated time for flow cytometry analysis. 12 h later nocodazole was added at 0.1 μg/ml final concentration. (B) Synchronized cells were taken at the indicated time and total  extracts were prepared. Equal quantities of protein were subjected to SDS-PAGE in 5% acrylamide gels and immunoblotted with α-BRG1  and α-hBRM antibodies. Phosphorylated and unphosphorylated BRG1 and hBRM are indicated. (C) Synchronized cells were taken at  the indicated time and fractionated as described in Fig. 2. Cytoplasmic (Cy), chromatin (Chr), and nuclear matrix (NM) fractions were  electrophoresed in 5% acrylamide gels and immunoblotted with α-BRG1 and α-hBRM antibodies.
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Figure 5: Association of BRG1 and hBRM with the nuclear matrix in synchronized HeLa cells. (A) HeLa Cells were synchronized by treatment with thymidine for 40 h. After release of the block (t = 0), samples were taken at the indicated time for flow cytometry analysis. 12 h later nocodazole was added at 0.1 μg/ml final concentration. (B) Synchronized cells were taken at the indicated time and total extracts were prepared. Equal quantities of protein were subjected to SDS-PAGE in 5% acrylamide gels and immunoblotted with α-BRG1 and α-hBRM antibodies. Phosphorylated and unphosphorylated BRG1 and hBRM are indicated. (C) Synchronized cells were taken at the indicated time and fractionated as described in Fig. 2. Cytoplasmic (Cy), chromatin (Chr), and nuclear matrix (NM) fractions were electrophoresed in 5% acrylamide gels and immunoblotted with α-BRG1 and α-hBRM antibodies.
Mentions: We have recently demonstrated that hBRM and BRG1 are phosphorylated in mitotic cells. The phosphorylated forms of the proteins are localized in the entire cell volume with exclusion from the condensed chromosomes (Muchardt et al., 1996). To determine more accurately the moment at which phosphorylation occurs and to pinpoint whether a correlation exists between the nature of the nuclear association of the hSWI/SNF complex and the position in the cell cycle, we have fractionated nuclei of HeLa cells synchronized at various stages of the cycle. Exponentially growing HeLa cells were blocked with thymidine (S-phase arrest) for 40 h, and then released from the block. Flow cytometry analysis revealed that after 12 h ∼95% of the cells contained a 4 N chromosomal content (Fig. 5 A). However, only ∼15% of the cells had entered mitosis, as determined by phase-contrast microscopy. At that time, nocodazole was added to the medium to block the cells in mitosis. Samples were taken at different times and total extracts were prepared. Alternatively, cells were subjected to the DNase I–high salt fractionation protocol described above. As shown in Fig. 5 B, both BRG1 and hBRM were phosphorylated at the time when cells entered mitosis but not in S and G2 phases. Both BRG1 and hBRM remained associated with the nuclear matrix during G1, S, and G2 phases (Fig. 5 C). However, all of the signal appeared in the cytoplasmic fraction in mitotic cells, consistent with the breakdown of the nuclear structure at mitosis and the exclusion of hBRM and BRG1 from the condensed chromatin as previously published (Muchardt et al., 1996).

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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