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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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The product of the  Hmg1 gene localizes to the nucleus. NIH 3T3 fibroblasts stably transfected with the  pHMG1tag plasmid were fixed  and stained simultaneously  with (A) Hoechst 33258, (B)  anti-HMG1 antibody chIPAB, and (C) monoclonal antibody 12CA5 recognizing the  HA epitope. (D) Structure of  the Hmg1-tag gene. The mouse  gene Hmg1, which codes for  protein HMG1, is transcribed  under the control of its own  strong promoter/enhancer. Exons are numbered. Black boxes  represent untranslated sequences  and white boxes translated sequences. Plasmid pmHMG1  was modified by the insertion  of 27 bp coding for the HA  epitope (bold and underlined)  immediately after the ATG  codon for the first methionine  of HMG1 and in frame with the  rest of the protein. A stable  clone (c47) expressing HMG1tag  approximately to the same  level of unmodified HMG1 was selected. (E) Western blotting of whole cell extracts from wild-type fibroblasts (lanes 3T3) and from the  stable transfected cells (lanes c47). The anti-HMG1 antibody chWB-AB (left) recognizes in the same way HMG1 and HMG1tag; the  monoclonal antibody 12CA5 recognizes the HA epitope (right). Protein HMG1tag runs slightly slower than wild-type HMG1 in tricine– SDS-PAGE because of the addition of nine amino acids.
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Figure 2: The product of the Hmg1 gene localizes to the nucleus. NIH 3T3 fibroblasts stably transfected with the pHMG1tag plasmid were fixed and stained simultaneously with (A) Hoechst 33258, (B) anti-HMG1 antibody chIPAB, and (C) monoclonal antibody 12CA5 recognizing the HA epitope. (D) Structure of the Hmg1-tag gene. The mouse gene Hmg1, which codes for protein HMG1, is transcribed under the control of its own strong promoter/enhancer. Exons are numbered. Black boxes represent untranslated sequences and white boxes translated sequences. Plasmid pmHMG1 was modified by the insertion of 27 bp coding for the HA epitope (bold and underlined) immediately after the ATG codon for the first methionine of HMG1 and in frame with the rest of the protein. A stable clone (c47) expressing HMG1tag approximately to the same level of unmodified HMG1 was selected. (E) Western blotting of whole cell extracts from wild-type fibroblasts (lanes 3T3) and from the stable transfected cells (lanes c47). The anti-HMG1 antibody chWB-AB (left) recognizes in the same way HMG1 and HMG1tag; the monoclonal antibody 12CA5 recognizes the HA epitope (right). Protein HMG1tag runs slightly slower than wild-type HMG1 in tricine– SDS-PAGE because of the addition of nine amino acids.

Mentions: HMG1 is a member of a protein family including HMG2 and ∼80 HMG1-related sequences (Ferrari et al., 1994). To exclude the possibility that our antibodies could cross react with proteins closely related to HMG1, we modified the cloned Hmg1 gene by adding a sequence coding for a nonapeptide from influenza hemagglutinin. The modification does not alter the exon–intron organization of the gene nor its 5′ untranscribed region (Fig. 2 D). We obtained several NIH 3T3 clones stably transformed with the tagged gene, one of which (c47) expressed similar amounts of HMG1 and HMG1tag (Fig. 2 E). The anti-HA mAb does not stain wild-type NIH 3T3 fibroblasts but brightly stains the nucleus of transformed cells (Fig. 2 C).


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)

The product of the  Hmg1 gene localizes to the nucleus. NIH 3T3 fibroblasts stably transfected with the  pHMG1tag plasmid were fixed  and stained simultaneously  with (A) Hoechst 33258, (B)  anti-HMG1 antibody chIPAB, and (C) monoclonal antibody 12CA5 recognizing the  HA epitope. (D) Structure of  the Hmg1-tag gene. The mouse  gene Hmg1, which codes for  protein HMG1, is transcribed  under the control of its own  strong promoter/enhancer. Exons are numbered. Black boxes  represent untranslated sequences  and white boxes translated sequences. Plasmid pmHMG1  was modified by the insertion  of 27 bp coding for the HA  epitope (bold and underlined)  immediately after the ATG  codon for the first methionine  of HMG1 and in frame with the  rest of the protein. A stable  clone (c47) expressing HMG1tag  approximately to the same  level of unmodified HMG1 was selected. (E) Western blotting of whole cell extracts from wild-type fibroblasts (lanes 3T3) and from the  stable transfected cells (lanes c47). The anti-HMG1 antibody chWB-AB (left) recognizes in the same way HMG1 and HMG1tag; the  monoclonal antibody 12CA5 recognizes the HA epitope (right). Protein HMG1tag runs slightly slower than wild-type HMG1 in tricine– SDS-PAGE because of the addition of nine amino acids.
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Related In: Results  -  Collection

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Figure 2: The product of the Hmg1 gene localizes to the nucleus. NIH 3T3 fibroblasts stably transfected with the pHMG1tag plasmid were fixed and stained simultaneously with (A) Hoechst 33258, (B) anti-HMG1 antibody chIPAB, and (C) monoclonal antibody 12CA5 recognizing the HA epitope. (D) Structure of the Hmg1-tag gene. The mouse gene Hmg1, which codes for protein HMG1, is transcribed under the control of its own strong promoter/enhancer. Exons are numbered. Black boxes represent untranslated sequences and white boxes translated sequences. Plasmid pmHMG1 was modified by the insertion of 27 bp coding for the HA epitope (bold and underlined) immediately after the ATG codon for the first methionine of HMG1 and in frame with the rest of the protein. A stable clone (c47) expressing HMG1tag approximately to the same level of unmodified HMG1 was selected. (E) Western blotting of whole cell extracts from wild-type fibroblasts (lanes 3T3) and from the stable transfected cells (lanes c47). The anti-HMG1 antibody chWB-AB (left) recognizes in the same way HMG1 and HMG1tag; the monoclonal antibody 12CA5 recognizes the HA epitope (right). Protein HMG1tag runs slightly slower than wild-type HMG1 in tricine– SDS-PAGE because of the addition of nine amino acids.
Mentions: HMG1 is a member of a protein family including HMG2 and ∼80 HMG1-related sequences (Ferrari et al., 1994). To exclude the possibility that our antibodies could cross react with proteins closely related to HMG1, we modified the cloned Hmg1 gene by adding a sequence coding for a nonapeptide from influenza hemagglutinin. The modification does not alter the exon–intron organization of the gene nor its 5′ untranscribed region (Fig. 2 D). We obtained several NIH 3T3 clones stably transformed with the tagged gene, one of which (c47) expressed similar amounts of HMG1 and HMG1tag (Fig. 2 E). The anti-HA mAb does not stain wild-type NIH 3T3 fibroblasts but brightly stains the nucleus of transformed cells (Fig. 2 C).

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