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A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome.

Chadwick BP, Willard HF - J. Cell Biol. (2001)

Bottom Line: In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout.In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes.The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Case Western Reserve University School of Medicine and Center for Human Genetics and Research Institute, University Hospitals of Cleveland, Cleveland, Ohio 44106-4955, USA.

ABSTRACT
Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

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Nuclear distribution of H2A-Bbd at interphase in primary female fibroblast cells showing the zone of exclusion around the Barr body and X chromosome, and the distribution of macroH2A in 46,XY and 47,XXX primary fibroblast cell nuclei. (a) Transfected female cell showing the nuclear distribution of a COOH-terminal GFP-tagged H2A-Bbd by indirect immunofluorescence (green). The region of exclusion is indicated with the white arrow. (a′) DAPI staining of the same nucleus reveals the condensed Xi in the form of the Barr body at the periphery of the nucleus (white arrow). (b) Indirect immunofluorescence of a transfected female cell showing the nuclear distribution of a COOH-terminal myc-tagged H2A-Bbd (red, TR), stained with a anti–myc mAb followed by goat anti–mouse IgG conjugated with Texas red. The exclusion is indicated with a white arrow. (b′) The DAPI image of the same nucleus indicates the position of the Barr body. (c) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution of H2A-Bbd by indirect immunofluorescence (green, FITC) merged with the FISH signals for a human X alpha satellite probe (orange, rhodamine). One of the two X centromere signals is located within a deficient region and is indicated with a white arrow. (c′) The DAPI image of the same nucleus indicates the location of the X alpha satellite FISH probe that is contained within the H2A-Bbd exclusion zone. (d) Transfected female cell showing the even nuclear distribution of a COOH-terminal myc-epitope–tagged H2B construct by indirect immunofluorescence (green, FITC) superimposed with two X alpha satellite FISH signals (orange, rhodamine). Neither X chromosome is located within a region deficient in H2B staining. (d′) DAPI stain of the same nucleus is shown. (e) Transfected interphase male cell showing the nuclear distribution of a COOH-terminal myc-epitope–tagged macroH2A construct by indirect immunofluorescence (green, FITC). (e′) The location of the single X chromosome is shown merged with the DAPI stain of the same nucleus (orange, rhodamine). No MCBs characteristic of an Xi can be seen in the nucleus. (f) Indirect immunofluorescence of a female interphase triple X cell transfected with macroH2A showing the nuclear location of two MCBs (green, FITC) indicated by white arrows. (f′) The DAPI stain of the same nucleus merged with the X alpha satellite FISH (orange, rhodamine) shows two X signals to be coincident with the MCB indicative of Xi's. The third X chromosome is not associated with an MCB.
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Figure 2: Nuclear distribution of H2A-Bbd at interphase in primary female fibroblast cells showing the zone of exclusion around the Barr body and X chromosome, and the distribution of macroH2A in 46,XY and 47,XXX primary fibroblast cell nuclei. (a) Transfected female cell showing the nuclear distribution of a COOH-terminal GFP-tagged H2A-Bbd by indirect immunofluorescence (green). The region of exclusion is indicated with the white arrow. (a′) DAPI staining of the same nucleus reveals the condensed Xi in the form of the Barr body at the periphery of the nucleus (white arrow). (b) Indirect immunofluorescence of a transfected female cell showing the nuclear distribution of a COOH-terminal myc-tagged H2A-Bbd (red, TR), stained with a anti–myc mAb followed by goat anti–mouse IgG conjugated with Texas red. The exclusion is indicated with a white arrow. (b′) The DAPI image of the same nucleus indicates the position of the Barr body. (c) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution of H2A-Bbd by indirect immunofluorescence (green, FITC) merged with the FISH signals for a human X alpha satellite probe (orange, rhodamine). One of the two X centromere signals is located within a deficient region and is indicated with a white arrow. (c′) The DAPI image of the same nucleus indicates the location of the X alpha satellite FISH probe that is contained within the H2A-Bbd exclusion zone. (d) Transfected female cell showing the even nuclear distribution of a COOH-terminal myc-epitope–tagged H2B construct by indirect immunofluorescence (green, FITC) superimposed with two X alpha satellite FISH signals (orange, rhodamine). Neither X chromosome is located within a region deficient in H2B staining. (d′) DAPI stain of the same nucleus is shown. (e) Transfected interphase male cell showing the nuclear distribution of a COOH-terminal myc-epitope–tagged macroH2A construct by indirect immunofluorescence (green, FITC). (e′) The location of the single X chromosome is shown merged with the DAPI stain of the same nucleus (orange, rhodamine). No MCBs characteristic of an Xi can be seen in the nucleus. (f) Indirect immunofluorescence of a female interphase triple X cell transfected with macroH2A showing the nuclear location of two MCBs (green, FITC) indicated by white arrows. (f′) The DAPI stain of the same nucleus merged with the X alpha satellite FISH (orange, rhodamine) shows two X signals to be coincident with the MCB indicative of Xi's. The third X chromosome is not associated with an MCB.

Mentions: To investigate the subcellular localization of the protein, a GFP fusion to the COOH terminus of the histone variant was constructed and transfected into primary human fibroblasts. In 100% of transfected 46,XY cells, a uniformly distributed GFP signal was observed in the nucleus. However, in female cells, counterstaining of the nucleus with DAPI revealed a clear excluded region in the distribution of the protein at the periphery of the nucleus, in a region corresponding to the Barr body (Fig. 2 a). To address the possibility that the protein was being excluded from the Barr body because of the large GFP fusion, we performed similar experiments using a myc-tagged version of the protein. The myc-tagged protein also localized to the nucleus and again, an exclusion zone was observed where the Barr body was found (Fig. 2 b). We have named the protein H2A-Bbd, for Histone H2A variant, Barr-body deficient, to reflect this pattern.


A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome.

Chadwick BP, Willard HF - J. Cell Biol. (2001)

Nuclear distribution of H2A-Bbd at interphase in primary female fibroblast cells showing the zone of exclusion around the Barr body and X chromosome, and the distribution of macroH2A in 46,XY and 47,XXX primary fibroblast cell nuclei. (a) Transfected female cell showing the nuclear distribution of a COOH-terminal GFP-tagged H2A-Bbd by indirect immunofluorescence (green). The region of exclusion is indicated with the white arrow. (a′) DAPI staining of the same nucleus reveals the condensed Xi in the form of the Barr body at the periphery of the nucleus (white arrow). (b) Indirect immunofluorescence of a transfected female cell showing the nuclear distribution of a COOH-terminal myc-tagged H2A-Bbd (red, TR), stained with a anti–myc mAb followed by goat anti–mouse IgG conjugated with Texas red. The exclusion is indicated with a white arrow. (b′) The DAPI image of the same nucleus indicates the position of the Barr body. (c) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution of H2A-Bbd by indirect immunofluorescence (green, FITC) merged with the FISH signals for a human X alpha satellite probe (orange, rhodamine). One of the two X centromere signals is located within a deficient region and is indicated with a white arrow. (c′) The DAPI image of the same nucleus indicates the location of the X alpha satellite FISH probe that is contained within the H2A-Bbd exclusion zone. (d) Transfected female cell showing the even nuclear distribution of a COOH-terminal myc-epitope–tagged H2B construct by indirect immunofluorescence (green, FITC) superimposed with two X alpha satellite FISH signals (orange, rhodamine). Neither X chromosome is located within a region deficient in H2B staining. (d′) DAPI stain of the same nucleus is shown. (e) Transfected interphase male cell showing the nuclear distribution of a COOH-terminal myc-epitope–tagged macroH2A construct by indirect immunofluorescence (green, FITC). (e′) The location of the single X chromosome is shown merged with the DAPI stain of the same nucleus (orange, rhodamine). No MCBs characteristic of an Xi can be seen in the nucleus. (f) Indirect immunofluorescence of a female interphase triple X cell transfected with macroH2A showing the nuclear location of two MCBs (green, FITC) indicated by white arrows. (f′) The DAPI stain of the same nucleus merged with the X alpha satellite FISH (orange, rhodamine) shows two X signals to be coincident with the MCB indicative of Xi's. The third X chromosome is not associated with an MCB.
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Related In: Results  -  Collection

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Figure 2: Nuclear distribution of H2A-Bbd at interphase in primary female fibroblast cells showing the zone of exclusion around the Barr body and X chromosome, and the distribution of macroH2A in 46,XY and 47,XXX primary fibroblast cell nuclei. (a) Transfected female cell showing the nuclear distribution of a COOH-terminal GFP-tagged H2A-Bbd by indirect immunofluorescence (green). The region of exclusion is indicated with the white arrow. (a′) DAPI staining of the same nucleus reveals the condensed Xi in the form of the Barr body at the periphery of the nucleus (white arrow). (b) Indirect immunofluorescence of a transfected female cell showing the nuclear distribution of a COOH-terminal myc-tagged H2A-Bbd (red, TR), stained with a anti–myc mAb followed by goat anti–mouse IgG conjugated with Texas red. The exclusion is indicated with a white arrow. (b′) The DAPI image of the same nucleus indicates the position of the Barr body. (c) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution of H2A-Bbd by indirect immunofluorescence (green, FITC) merged with the FISH signals for a human X alpha satellite probe (orange, rhodamine). One of the two X centromere signals is located within a deficient region and is indicated with a white arrow. (c′) The DAPI image of the same nucleus indicates the location of the X alpha satellite FISH probe that is contained within the H2A-Bbd exclusion zone. (d) Transfected female cell showing the even nuclear distribution of a COOH-terminal myc-epitope–tagged H2B construct by indirect immunofluorescence (green, FITC) superimposed with two X alpha satellite FISH signals (orange, rhodamine). Neither X chromosome is located within a region deficient in H2B staining. (d′) DAPI stain of the same nucleus is shown. (e) Transfected interphase male cell showing the nuclear distribution of a COOH-terminal myc-epitope–tagged macroH2A construct by indirect immunofluorescence (green, FITC). (e′) The location of the single X chromosome is shown merged with the DAPI stain of the same nucleus (orange, rhodamine). No MCBs characteristic of an Xi can be seen in the nucleus. (f) Indirect immunofluorescence of a female interphase triple X cell transfected with macroH2A showing the nuclear location of two MCBs (green, FITC) indicated by white arrows. (f′) The DAPI stain of the same nucleus merged with the X alpha satellite FISH (orange, rhodamine) shows two X signals to be coincident with the MCB indicative of Xi's. The third X chromosome is not associated with an MCB.
Mentions: To investigate the subcellular localization of the protein, a GFP fusion to the COOH terminus of the histone variant was constructed and transfected into primary human fibroblasts. In 100% of transfected 46,XY cells, a uniformly distributed GFP signal was observed in the nucleus. However, in female cells, counterstaining of the nucleus with DAPI revealed a clear excluded region in the distribution of the protein at the periphery of the nucleus, in a region corresponding to the Barr body (Fig. 2 a). To address the possibility that the protein was being excluded from the Barr body because of the large GFP fusion, we performed similar experiments using a myc-tagged version of the protein. The myc-tagged protein also localized to the nucleus and again, an exclusion zone was observed where the Barr body was found (Fig. 2 b). We have named the protein H2A-Bbd, for Histone H2A variant, Barr-body deficient, to reflect this pattern.

Bottom Line: In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout.In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes.The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Case Western Reserve University School of Medicine and Center for Human Genetics and Research Institute, University Hospitals of Cleveland, Cleveland, Ohio 44106-4955, USA.

ABSTRACT
Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

Show MeSH
Related in: MedlinePlus