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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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(A) Reactivity of  the various anti-HMG1 antibodies used in this study. For  each antibody preparation  the species of origin is indicated (m, mouse; ch,  chicken), and the ability (+)  or inability (−) to recognize  boxes A and B of HMG1 by  Western blotting (denatured) or by immunoprecipitation (native) is also shown.  The notation ± indicates that  recovery of the HMG1bA  polypeptide by immunoprecipitation is <5% under the  conditions indicated under  Materials and Methods. (B)  Reactivity of the chWB-AB  antibody in Western blots.  Whole NIH 3T3 cells were  lysed by addition of SDSPAGE loading buffer, and  10 μg of total protein was  loaded on a 10% tricine–SDS– polyacrylamide gel (lane  3T3), alongside 20 ng of purified recombinant HMG1bA  polypeptide (lane bA) or 20 ng of purified recombinant HMG1bB polypeptide (lane bB). (C and D) Anti-HMG1 antibodies stain the  cell nucleus. NIH 3T3 cells were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with 0.1% NP-40, and stained  with anti-recAtn (C) and Hoechst 33258 (D). (E and F) Localization of HMG1 by confocal microscopy. HeLa cells were fixed with  paraformaldehyde, permeabilized with 0.1% SDS, stained with mAP-bA antibody, and viewed in green fluorescence (E) or by phase  contrast microscopy (F). Bars, 10 μm.
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Figure 1: (A) Reactivity of the various anti-HMG1 antibodies used in this study. For each antibody preparation the species of origin is indicated (m, mouse; ch, chicken), and the ability (+) or inability (−) to recognize boxes A and B of HMG1 by Western blotting (denatured) or by immunoprecipitation (native) is also shown. The notation ± indicates that recovery of the HMG1bA polypeptide by immunoprecipitation is <5% under the conditions indicated under Materials and Methods. (B) Reactivity of the chWB-AB antibody in Western blots. Whole NIH 3T3 cells were lysed by addition of SDSPAGE loading buffer, and 10 μg of total protein was loaded on a 10% tricine–SDS– polyacrylamide gel (lane 3T3), alongside 20 ng of purified recombinant HMG1bA polypeptide (lane bA) or 20 ng of purified recombinant HMG1bB polypeptide (lane bB). (C and D) Anti-HMG1 antibodies stain the cell nucleus. NIH 3T3 cells were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with 0.1% NP-40, and stained with anti-recAtn (C) and Hoechst 33258 (D). (E and F) Localization of HMG1 by confocal microscopy. HeLa cells were fixed with paraformaldehyde, permeabilized with 0.1% SDS, stained with mAP-bA antibody, and viewed in green fluorescence (E) or by phase contrast microscopy (F). Bars, 10 μm.

Mentions: The four different antibody preparations have different reactivities against HMG1, as shown in Fig. 1 A. Reactivity against the native antigens was assessed by dot immunoblots or immunoprecipitation experiments (not shown); reactivity against the denatured antigens was assessed with Western blots (Fig. 1 B). In different cell lines (NIH 3T3 fibroblasts, HeLa and Jurkat cells, and mouse Schwann cells) each of the antibodies stained the nucleus (Fig. 1, C–F and results not shown). The result was the same with several fixation regimes and permeabilization agents. Significantly, the distribution of HMG1 and of AT-rich heterochromatin (revealed by Hoechst 33258 as spots of brighter fluorescence) do not correlate (Fig. 1, compare C with D). Confocal microscopy (Fig. 1, E and F) revealed a diffuse, finely punctate pattern of staining, with little or no HMG1 in the nucleoli.


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)

(A) Reactivity of  the various anti-HMG1 antibodies used in this study. For  each antibody preparation  the species of origin is indicated (m, mouse; ch,  chicken), and the ability (+)  or inability (−) to recognize  boxes A and B of HMG1 by  Western blotting (denatured) or by immunoprecipitation (native) is also shown.  The notation ± indicates that  recovery of the HMG1bA  polypeptide by immunoprecipitation is <5% under the  conditions indicated under  Materials and Methods. (B)  Reactivity of the chWB-AB  antibody in Western blots.  Whole NIH 3T3 cells were  lysed by addition of SDSPAGE loading buffer, and  10 μg of total protein was  loaded on a 10% tricine–SDS– polyacrylamide gel (lane  3T3), alongside 20 ng of purified recombinant HMG1bA  polypeptide (lane bA) or 20 ng of purified recombinant HMG1bB polypeptide (lane bB). (C and D) Anti-HMG1 antibodies stain the  cell nucleus. NIH 3T3 cells were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with 0.1% NP-40, and stained  with anti-recAtn (C) and Hoechst 33258 (D). (E and F) Localization of HMG1 by confocal microscopy. HeLa cells were fixed with  paraformaldehyde, permeabilized with 0.1% SDS, stained with mAP-bA antibody, and viewed in green fluorescence (E) or by phase  contrast microscopy (F). Bars, 10 μm.
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Figure 1: (A) Reactivity of the various anti-HMG1 antibodies used in this study. For each antibody preparation the species of origin is indicated (m, mouse; ch, chicken), and the ability (+) or inability (−) to recognize boxes A and B of HMG1 by Western blotting (denatured) or by immunoprecipitation (native) is also shown. The notation ± indicates that recovery of the HMG1bA polypeptide by immunoprecipitation is <5% under the conditions indicated under Materials and Methods. (B) Reactivity of the chWB-AB antibody in Western blots. Whole NIH 3T3 cells were lysed by addition of SDSPAGE loading buffer, and 10 μg of total protein was loaded on a 10% tricine–SDS– polyacrylamide gel (lane 3T3), alongside 20 ng of purified recombinant HMG1bA polypeptide (lane bA) or 20 ng of purified recombinant HMG1bB polypeptide (lane bB). (C and D) Anti-HMG1 antibodies stain the cell nucleus. NIH 3T3 cells were grown on glass coverslips, fixed with paraformaldehyde, permeabilized with 0.1% NP-40, and stained with anti-recAtn (C) and Hoechst 33258 (D). (E and F) Localization of HMG1 by confocal microscopy. HeLa cells were fixed with paraformaldehyde, permeabilized with 0.1% SDS, stained with mAP-bA antibody, and viewed in green fluorescence (E) or by phase contrast microscopy (F). Bars, 10 μm.
Mentions: The four different antibody preparations have different reactivities against HMG1, as shown in Fig. 1 A. Reactivity against the native antigens was assessed by dot immunoblots or immunoprecipitation experiments (not shown); reactivity against the denatured antigens was assessed with Western blots (Fig. 1 B). In different cell lines (NIH 3T3 fibroblasts, HeLa and Jurkat cells, and mouse Schwann cells) each of the antibodies stained the nucleus (Fig. 1, C–F and results not shown). The result was the same with several fixation regimes and permeabilization agents. Significantly, the distribution of HMG1 and of AT-rich heterochromatin (revealed by Hoechst 33258 as spots of brighter fluorescence) do not correlate (Fig. 1, compare C with D). Confocal microscopy (Fig. 1, E and F) revealed a diffuse, finely punctate pattern of staining, with little or no HMG1 in the nucleoli.

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