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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, Universit√° 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 protein is not associated to mitotic condensed chromosomes. Dividing NIH 3T3 fibroblasts were fixed and stained for  HMG1 with antibody chIP-AB (top row, green fluorescence) and for DNA with Hoechst 33258 (bottom row, blue fluorescence). Representative cells at different stages during mitosis: prophase (A), metaphase (B), anaphase (C), and telophase (D). After the breakdown  of the nuclear membrane, HMG1 diffuses throughout the cytoplasm, and the pattern of green fluorescence corresponds to the shape of  the cell. However, fluorescence from HMG1 is clearly reduced in correspondence to the volume occupied by condensed chromosomes  (compare top and bottom images), indicating that HMG1 is not associated with DNA during mitosis. After cell division and the reformation of nuclear membranes (D), the majority of HMG1 colocalizes with DNA, but some is still found in the cytoplasm, suggesting  that the protein is being concentrated in the nuclei by passage through the nuclear membrane.
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Figure 4: HMG1 protein is not associated to mitotic condensed chromosomes. Dividing NIH 3T3 fibroblasts were fixed and stained for HMG1 with antibody chIP-AB (top row, green fluorescence) and for DNA with Hoechst 33258 (bottom row, blue fluorescence). Representative cells at different stages during mitosis: prophase (A), metaphase (B), anaphase (C), and telophase (D). After the breakdown of the nuclear membrane, HMG1 diffuses throughout the cytoplasm, and the pattern of green fluorescence corresponds to the shape of the cell. However, fluorescence from HMG1 is clearly reduced in correspondence to the volume occupied by condensed chromosomes (compare top and bottom images), indicating that HMG1 is not associated with DNA during mitosis. After cell division and the reformation of nuclear membranes (D), the majority of HMG1 colocalizes with DNA, but some is still found in the cytoplasm, suggesting that the protein is being concentrated in the nuclei by passage through the nuclear membrane.

Mentions: To further confirm the association of HMG1 with nucleosomes, we then probed whether HMG1 is an integral component of condensed chromosomes, like its proposed Drosophila homolog, HMG-D (Ner and Travers, 1994), and histone H1 (Breneman et al., 1993). Unexpectedly, in fibroblasts undergoing mitosis our anti-HMG1 antibodies stained the cytoplasm in a diffuse way (Fig. 4 and results not shown), while the chromosomes appear as dark areas, indicating that HMG1 is displaced from condensed chromatin. As a control for the accessibility of mitotic chromosomes to antibodies, the same cells were stained with the monoclonal IgM antibody HBC-7 directed against histone H2B (Whitfield et al., 1986) and with a polyclonal rabbit antibody directed against histone H1. In contrast to the anti-HMG1 antibodies, the antibodies against core and linker histones brightly stained the condensed chromosomes (Fig. 5).


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 protein is not associated to mitotic condensed chromosomes. Dividing NIH 3T3 fibroblasts were fixed and stained for  HMG1 with antibody chIP-AB (top row, green fluorescence) and for DNA with Hoechst 33258 (bottom row, blue fluorescence). Representative cells at different stages during mitosis: prophase (A), metaphase (B), anaphase (C), and telophase (D). After the breakdown  of the nuclear membrane, HMG1 diffuses throughout the cytoplasm, and the pattern of green fluorescence corresponds to the shape of  the cell. However, fluorescence from HMG1 is clearly reduced in correspondence to the volume occupied by condensed chromosomes  (compare top and bottom images), indicating that HMG1 is not associated with DNA during mitosis. After cell division and the reformation of nuclear membranes (D), the majority of HMG1 colocalizes with DNA, but some is still found in the cytoplasm, suggesting  that the protein is being concentrated in the nuclei by passage through the nuclear membrane.
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Related In: Results  -  Collection

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Figure 4: HMG1 protein is not associated to mitotic condensed chromosomes. Dividing NIH 3T3 fibroblasts were fixed and stained for HMG1 with antibody chIP-AB (top row, green fluorescence) and for DNA with Hoechst 33258 (bottom row, blue fluorescence). Representative cells at different stages during mitosis: prophase (A), metaphase (B), anaphase (C), and telophase (D). After the breakdown of the nuclear membrane, HMG1 diffuses throughout the cytoplasm, and the pattern of green fluorescence corresponds to the shape of the cell. However, fluorescence from HMG1 is clearly reduced in correspondence to the volume occupied by condensed chromosomes (compare top and bottom images), indicating that HMG1 is not associated with DNA during mitosis. After cell division and the reformation of nuclear membranes (D), the majority of HMG1 colocalizes with DNA, but some is still found in the cytoplasm, suggesting that the protein is being concentrated in the nuclei by passage through the nuclear membrane.
Mentions: To further confirm the association of HMG1 with nucleosomes, we then probed whether HMG1 is an integral component of condensed chromosomes, like its proposed Drosophila homolog, HMG-D (Ner and Travers, 1994), and histone H1 (Breneman et al., 1993). Unexpectedly, in fibroblasts undergoing mitosis our anti-HMG1 antibodies stained the cytoplasm in a diffuse way (Fig. 4 and results not shown), while the chromosomes appear as dark areas, indicating that HMG1 is displaced from condensed chromatin. As a control for the accessibility of mitotic chromosomes to antibodies, the same cells were stained with the monoclonal IgM antibody HBC-7 directed against histone H2B (Whitfield et al., 1986) and with a polyclonal rabbit antibody directed against histone H1. In contrast to the anti-HMG1 antibodies, the antibodies against core and linker histones brightly stained the condensed chromosomes (Fig. 5).

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, Universit√° 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