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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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In situ extraction of nuclear  matrix. (A) Immunofluorescent  staining of nonextracted HeLa cells  with α-BRG1, α-hBRM, and α-lamin  B antibodies. (B) Immunofluorescent staining pattern of in situ prepared nuclear matrices with α-BRG1,  α-hBRM, and α-lamin B antibodies. Microscopy and photography  parameters were standardized for  all of the images. Bar, 10 μm.
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Figure 3: In situ extraction of nuclear matrix. (A) Immunofluorescent staining of nonextracted HeLa cells with α-BRG1, α-hBRM, and α-lamin B antibodies. (B) Immunofluorescent staining pattern of in situ prepared nuclear matrices with α-BRG1, α-hBRM, and α-lamin B antibodies. Microscopy and photography parameters were standardized for all of the images. Bar, 10 μm.

Mentions: To investigate whether the distribution of hBRM and BRG1 proteins is similar in whole nuclei and in nuclear matrices, we used α-hBRM and α-BRG1 antibodies in indirect immunofluorescence analysis of the nuclear material remaining after in situ sequential DNase I and high salt extraction. As shown in Fig. 3, the labeling pattern was similar for both proteins before and after extraction, but the intensity of the signal decreased in extracted nuclei. As expected, the DAPI signal disappeared completely in the nuclear matrix preparations (Fig. 3 B). α-lamin B antibodies gave a strong signal in the nuclear lamina and a much weaker, diffuse signal in the nucleoplasm. The intensity of the signal was similar in whole nuclei and in extracted nuclei; however, lamina of extracted nuclei appeared slightly deformed or broken, probably as a result of the osmotic shock occurring during the extraction procedure (Fig. 3 B).


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)

In situ extraction of nuclear  matrix. (A) Immunofluorescent  staining of nonextracted HeLa cells  with α-BRG1, α-hBRM, and α-lamin  B antibodies. (B) Immunofluorescent staining pattern of in situ prepared nuclear matrices with α-BRG1,  α-hBRM, and α-lamin B antibodies. Microscopy and photography  parameters were standardized for  all of the images. Bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139781&req=5

Figure 3: In situ extraction of nuclear matrix. (A) Immunofluorescent staining of nonextracted HeLa cells with α-BRG1, α-hBRM, and α-lamin B antibodies. (B) Immunofluorescent staining pattern of in situ prepared nuclear matrices with α-BRG1, α-hBRM, and α-lamin B antibodies. Microscopy and photography parameters were standardized for all of the images. Bar, 10 μm.
Mentions: To investigate whether the distribution of hBRM and BRG1 proteins is similar in whole nuclei and in nuclear matrices, we used α-hBRM and α-BRG1 antibodies in indirect immunofluorescence analysis of the nuclear material remaining after in situ sequential DNase I and high salt extraction. As shown in Fig. 3, the labeling pattern was similar for both proteins before and after extraction, but the intensity of the signal decreased in extracted nuclei. As expected, the DAPI signal disappeared completely in the nuclear matrix preparations (Fig. 3 B). α-lamin B antibodies gave a strong signal in the nuclear lamina and a much weaker, diffuse signal in the nucleoplasm. The intensity of the signal was similar in whole nuclei and in extracted nuclei; however, lamina of extracted nuclei appeared slightly deformed or broken, probably as a result of the osmotic shock occurring during the extraction procedure (Fig. 3 B).

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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