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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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Active chromatin is enriched in hSWI/SNF components. 107 nuclei were subjected to partial digestion by micrococcal nuclease for the indicated times and separated into supernatant (S1) and pellet. The pellet was extracted with EDTA and  centrifuged to yield supernatant (S2) and pellet (P) chromatin  fractions. (A) DNA from each fraction was electrophoresed in 1%  agarose gels and stained with ethidium bromide. M, 100-bp standards. (B) An equivalent volume of each fraction was subjected to  SDS-PAGE and immunoblotted with α-BRG1, α-hBRM, α-SNF5,  and pol 3/3 (anti-RNAP II-LS) antibodies.
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Figure 4: Active chromatin is enriched in hSWI/SNF components. 107 nuclei were subjected to partial digestion by micrococcal nuclease for the indicated times and separated into supernatant (S1) and pellet. The pellet was extracted with EDTA and centrifuged to yield supernatant (S2) and pellet (P) chromatin fractions. (A) DNA from each fraction was electrophoresed in 1% agarose gels and stained with ethidium bromide. M, 100-bp standards. (B) An equivalent volume of each fraction was subjected to SDS-PAGE and immunoblotted with α-BRG1, α-hBRM, α-SNF5, and pol 3/3 (anti-RNAP II-LS) antibodies.

Mentions: In the previously described nuclear matrix isolation protocol, >95% of chromatin was solubilized in a single fraction. To try to partition between active and inactive chromatin, we have used an established fractionation procedure in which nuclei are subjected to mild digestion with micrococcal nuclease and subsequent extraction with EDTA (Huang and Garrard, 1989; Rose and Garrard, 1984). After nuclease treatment of HeLa cell nuclei for 1, 2, or 4 min and centrifugation, the supernatant fraction S1 contained between 1% and 8% of the total cellular DNA. The pellet was then resuspended in 2 mM EDTA, recentrifuged, and separated into a second supernatant fraction S2 and a pellet P, which contained ∼80% and 15% of the total DNA, respectively. As shown by DNA analysis in agarose gel, the S1 and S2 fractions represent differences in the nuclease accessibility of chromatin (Fig. 4 A). Fraction S1 is mainly composed of mononucleosomal-sized DNA fragments, whereas the S2 fraction contained a typical nucleosomal ladder of DNA fragments. The P fraction contained heterogeneous-sized DNA that remained bound to the nuclear scaffold after the fractionation. This fraction has also been called insoluble chromatin (Xu et al., 1986). It has been shown that S1 and P fractions are enriched in transcriptionally active DNA, whereas fraction S2 is depleted of transcribed sequences (Rose and Garrard, 1984). Aliquots of S1, S2, and P fractions were subjected to SDSPAGE followed by electrotransfer to nitrocellulose membranes and by immunoblot analysis. As shown in Fig. 4 B, BRG1, hBRM, and hSNF5 proteins were enriched in fractions S1 and P with respect to fraction S2. This enrichment appears to be higher for hBRM and hSNF5 than for BRG1 (around two- to threefold for BRG1, 10–15-fold for hBRM, and 20–30-fold for SNF5). Since fraction S2 contained the major part of the DNA, abundance of the hSWI/SNF proteins including BRG1, relative to DNA, was very low in this fraction. These data suggest that the hSWI/SNF complex is mostly concentrated in regions highly sensitive to micrococcal nuclease and in the fraction of the transcriptionally active chromatin that remains bound to the nuclear pellet after digestion. On the contrary, RNAP II was only found in fraction P, consistent with reports that demonstrate that transcriptional activity is associated with an insoluble phase of the nucleus (Jackson and Cook, 1985). Although at first view contradictory, this result is compatible with the fact that RNAP II was found in the soluble fraction in the experiments described in Fig. 2. RNAP II is extractable from the nucleus with the high salt concentration used in the nuclear matrix isolation protocol, but not with the low ionic strength buffers used in the chromatin fractionation protocol.


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)

Active chromatin is enriched in hSWI/SNF components. 107 nuclei were subjected to partial digestion by micrococcal nuclease for the indicated times and separated into supernatant (S1) and pellet. The pellet was extracted with EDTA and  centrifuged to yield supernatant (S2) and pellet (P) chromatin  fractions. (A) DNA from each fraction was electrophoresed in 1%  agarose gels and stained with ethidium bromide. M, 100-bp standards. (B) An equivalent volume of each fraction was subjected to  SDS-PAGE and immunoblotted with α-BRG1, α-hBRM, α-SNF5,  and pol 3/3 (anti-RNAP II-LS) antibodies.
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Related In: Results  -  Collection

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Figure 4: Active chromatin is enriched in hSWI/SNF components. 107 nuclei were subjected to partial digestion by micrococcal nuclease for the indicated times and separated into supernatant (S1) and pellet. The pellet was extracted with EDTA and centrifuged to yield supernatant (S2) and pellet (P) chromatin fractions. (A) DNA from each fraction was electrophoresed in 1% agarose gels and stained with ethidium bromide. M, 100-bp standards. (B) An equivalent volume of each fraction was subjected to SDS-PAGE and immunoblotted with α-BRG1, α-hBRM, α-SNF5, and pol 3/3 (anti-RNAP II-LS) antibodies.
Mentions: In the previously described nuclear matrix isolation protocol, >95% of chromatin was solubilized in a single fraction. To try to partition between active and inactive chromatin, we have used an established fractionation procedure in which nuclei are subjected to mild digestion with micrococcal nuclease and subsequent extraction with EDTA (Huang and Garrard, 1989; Rose and Garrard, 1984). After nuclease treatment of HeLa cell nuclei for 1, 2, or 4 min and centrifugation, the supernatant fraction S1 contained between 1% and 8% of the total cellular DNA. The pellet was then resuspended in 2 mM EDTA, recentrifuged, and separated into a second supernatant fraction S2 and a pellet P, which contained ∼80% and 15% of the total DNA, respectively. As shown by DNA analysis in agarose gel, the S1 and S2 fractions represent differences in the nuclease accessibility of chromatin (Fig. 4 A). Fraction S1 is mainly composed of mononucleosomal-sized DNA fragments, whereas the S2 fraction contained a typical nucleosomal ladder of DNA fragments. The P fraction contained heterogeneous-sized DNA that remained bound to the nuclear scaffold after the fractionation. This fraction has also been called insoluble chromatin (Xu et al., 1986). It has been shown that S1 and P fractions are enriched in transcriptionally active DNA, whereas fraction S2 is depleted of transcribed sequences (Rose and Garrard, 1984). Aliquots of S1, S2, and P fractions were subjected to SDSPAGE followed by electrotransfer to nitrocellulose membranes and by immunoblot analysis. As shown in Fig. 4 B, BRG1, hBRM, and hSNF5 proteins were enriched in fractions S1 and P with respect to fraction S2. This enrichment appears to be higher for hBRM and hSNF5 than for BRG1 (around two- to threefold for BRG1, 10–15-fold for hBRM, and 20–30-fold for SNF5). Since fraction S2 contained the major part of the DNA, abundance of the hSWI/SNF proteins including BRG1, relative to DNA, was very low in this fraction. These data suggest that the hSWI/SNF complex is mostly concentrated in regions highly sensitive to micrococcal nuclease and in the fraction of the transcriptionally active chromatin that remains bound to the nuclear pellet after digestion. On the contrary, RNAP II was only found in fraction P, consistent with reports that demonstrate that transcriptional activity is associated with an insoluble phase of the nucleus (Jackson and Cook, 1985). Although at first view contradictory, this result is compatible with the fact that RNAP II was found in the soluble fraction in the experiments described in Fig. 2. RNAP II is extractable from the nucleus with the high salt concentration used in the nuclear matrix isolation protocol, but not with the low ionic strength buffers used in the chromatin fractionation protocol.

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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