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High mobility group 1 protein is not stably associated with the chromosomes of somatic cells.

Falciola L, Spada F, Calogero S, Langst G, Voit R, Grummt I, Bianchi ME - J. Cell Biol. (1997)

Bottom Line: The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed.HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells.During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e di Biologia dei Microrganismi, Universitá di Milano, Italy.

ABSTRACT
High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

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HMG1 leaks out in  a similar way from both interphasic and mitotic permeabilized cells. NIH 3T3 fibroblasts were exposed overnight  to nocodazole, an inhibitor of  microtubule polymerization.  Cells that had entered M  phase could not proceed further and were detached from  their plastic substrate by  manual shaking (metaphase  cells). Cells that remained  adherent to the substrate after shaking (interphase cells)  were detached by treatment with trypsin. The two cell populations were checked for the presence of condensed chromosomes  (95% for metaphase cells; 2% for interphase cells). The cell suspensions were then exposed to 0.1% NP-40 and immediately centrifuged. Supernatants (lanes 1 and 3, S) and cell pellets (lanes 2  and 4, P) were analyzed by Western blotting with antibodies  against HMG1, histone H1, and protein HMG-I(Y).
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Figure 8: HMG1 leaks out in a similar way from both interphasic and mitotic permeabilized cells. NIH 3T3 fibroblasts were exposed overnight to nocodazole, an inhibitor of microtubule polymerization. Cells that had entered M phase could not proceed further and were detached from their plastic substrate by manual shaking (metaphase cells). Cells that remained adherent to the substrate after shaking (interphase cells) were detached by treatment with trypsin. The two cell populations were checked for the presence of condensed chromosomes (95% for metaphase cells; 2% for interphase cells). The cell suspensions were then exposed to 0.1% NP-40 and immediately centrifuged. Supernatants (lanes 1 and 3, S) and cell pellets (lanes 2 and 4, P) were analyzed by Western blotting with antibodies against HMG1, histone H1, and protein HMG-I(Y).

Mentions: We also compared directly the detergent-mediated release of HMG1 from metaphase and interphase cells. NIH 3T3 fibroblasts were treated with nocodazole, an inhibitor of microtubule assembly; mitotic cells were shaken off the dishes, while nonmitotic cells were detached by mild trypsin digestion (Martinez-Balbás et al., 1995). The two cell populations were then permeabilized with NP-40 and analyzed for protein retention by Western blotting (Fig. 8). In both mitotic and nonmitotic fibroblasts, histone H1 is retained in the cell pellets; in contrast, the vast majority of HMG1 is released into the medium. Moreover, HMG-I(Y), a different high mobility group protein which is a component of isolated condensed chromosomes (Saito and Laemmli, 1994) was retained after permeabilization in both mitotic and nonmitotic cells.


High mobility group 1 protein is not stably associated with the chromosomes of somatic cells.

Falciola L, Spada F, Calogero S, Langst G, Voit R, Grummt I, Bianchi ME - J. Cell Biol. (1997)

HMG1 leaks out in  a similar way from both interphasic and mitotic permeabilized cells. NIH 3T3 fibroblasts were exposed overnight  to nocodazole, an inhibitor of  microtubule polymerization.  Cells that had entered M  phase could not proceed further and were detached from  their plastic substrate by  manual shaking (metaphase  cells). Cells that remained  adherent to the substrate after shaking (interphase cells)  were detached by treatment with trypsin. The two cell populations were checked for the presence of condensed chromosomes  (95% for metaphase cells; 2% for interphase cells). The cell suspensions were then exposed to 0.1% NP-40 and immediately centrifuged. Supernatants (lanes 1 and 3, S) and cell pellets (lanes 2  and 4, P) were analyzed by Western blotting with antibodies  against HMG1, histone H1, and protein HMG-I(Y).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139855&req=5

Figure 8: HMG1 leaks out in a similar way from both interphasic and mitotic permeabilized cells. NIH 3T3 fibroblasts were exposed overnight to nocodazole, an inhibitor of microtubule polymerization. Cells that had entered M phase could not proceed further and were detached from their plastic substrate by manual shaking (metaphase cells). Cells that remained adherent to the substrate after shaking (interphase cells) were detached by treatment with trypsin. The two cell populations were checked for the presence of condensed chromosomes (95% for metaphase cells; 2% for interphase cells). The cell suspensions were then exposed to 0.1% NP-40 and immediately centrifuged. Supernatants (lanes 1 and 3, S) and cell pellets (lanes 2 and 4, P) were analyzed by Western blotting with antibodies against HMG1, histone H1, and protein HMG-I(Y).
Mentions: We also compared directly the detergent-mediated release of HMG1 from metaphase and interphase cells. NIH 3T3 fibroblasts were treated with nocodazole, an inhibitor of microtubule assembly; mitotic cells were shaken off the dishes, while nonmitotic cells were detached by mild trypsin digestion (Martinez-Balbás et al., 1995). The two cell populations were then permeabilized with NP-40 and analyzed for protein retention by Western blotting (Fig. 8). In both mitotic and nonmitotic fibroblasts, histone H1 is retained in the cell pellets; in contrast, the vast majority of HMG1 is released into the medium. Moreover, HMG-I(Y), a different high mobility group protein which is a component of isolated condensed chromosomes (Saito and Laemmli, 1994) was retained after permeabilization in both mitotic and nonmitotic cells.

Bottom Line: The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed.HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells.During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Genetica e di Biologia dei Microrganismi, Universitá di Milano, Italy.

ABSTRACT
High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

Show MeSH
Related in: MedlinePlus