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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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Female metaphase chromosomes from a H2A-Bbd–transfected 293 cell, counterstained for histone H4 acetylation at lysine-12. (a) DAPI stain of metaphase chromosomes merged with the FISH signal of an X alpha satellite probe (pink, Cy5). The positions of the two X chromosomes is indicated with white arrows. (b) Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes (red, rhodamine). The location of the two X chromosomes is shown with the white arrow, and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes is deficient for H2A-Bbd when compared with the other X chromosome and the surrounding autosomes. (c) Indirect immunofluorescence showing the distribution of H4Ac12 on metaphase chromosomes (green, FITC). The location of the X chromosomes is indicated with the white arrows and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes lacks H4Ac12 signal, indicative of the Xi. This is the same X shown in b to be deficient for H2A-Bbd. (d) Merge of the staining patterns of H2A-Bbd and H4Ac12. The orange color indicates a direct overlap of the H2A-Bbd and H4Ac12 signals.
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Figure 6: Female metaphase chromosomes from a H2A-Bbd–transfected 293 cell, counterstained for histone H4 acetylation at lysine-12. (a) DAPI stain of metaphase chromosomes merged with the FISH signal of an X alpha satellite probe (pink, Cy5). The positions of the two X chromosomes is indicated with white arrows. (b) Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes (red, rhodamine). The location of the two X chromosomes is shown with the white arrow, and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes is deficient for H2A-Bbd when compared with the other X chromosome and the surrounding autosomes. (c) Indirect immunofluorescence showing the distribution of H4Ac12 on metaphase chromosomes (green, FITC). The location of the X chromosomes is indicated with the white arrows and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes lacks H4Ac12 signal, indicative of the Xi. This is the same X shown in b to be deficient for H2A-Bbd. (d) Merge of the staining patterns of H2A-Bbd and H4Ac12. The orange color indicates a direct overlap of the H2A-Bbd and H4Ac12 signals.

Mentions: Metaphase spreads were prepared from H2A-Bbd–transfected 293 cells and coimmunostained for regions of H4 acetylation. The Xi has previously been shown to be hypoacetylated at H4 at metaphase (Jeppesen and Turner 1993), indicating acetylation status as a reliable marker for the Xi. While one X chromosome (the cytologically identifiable Xa) showed staining equivalent to the autosomes, additional X chromosomes in each spread lacked any H4Ac12 signal, identifying them as Xi's. As evaluated in >120 metaphase spreads, the same X chromosomes were also deficient for H2A-Bbd staining (Fig. 6). In no case was H2A-Bbd found to stain the underacetylated Xi chromosome. An overlay of both H2A-Bbd and H4Ac12 shows an identical pattern of distribution at metaphase (Fig. 6 d).


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)

Female metaphase chromosomes from a H2A-Bbd–transfected 293 cell, counterstained for histone H4 acetylation at lysine-12. (a) DAPI stain of metaphase chromosomes merged with the FISH signal of an X alpha satellite probe (pink, Cy5). The positions of the two X chromosomes is indicated with white arrows. (b) Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes (red, rhodamine). The location of the two X chromosomes is shown with the white arrow, and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes is deficient for H2A-Bbd when compared with the other X chromosome and the surrounding autosomes. (c) Indirect immunofluorescence showing the distribution of H4Ac12 on metaphase chromosomes (green, FITC). The location of the X chromosomes is indicated with the white arrows and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes lacks H4Ac12 signal, indicative of the Xi. This is the same X shown in b to be deficient for H2A-Bbd. (d) Merge of the staining patterns of H2A-Bbd and H4Ac12. The orange color indicates a direct overlap of the H2A-Bbd and H4Ac12 signals.
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Related In: Results  -  Collection

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Figure 6: Female metaphase chromosomes from a H2A-Bbd–transfected 293 cell, counterstained for histone H4 acetylation at lysine-12. (a) DAPI stain of metaphase chromosomes merged with the FISH signal of an X alpha satellite probe (pink, Cy5). The positions of the two X chromosomes is indicated with white arrows. (b) Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes (red, rhodamine). The location of the two X chromosomes is shown with the white arrow, and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes is deficient for H2A-Bbd when compared with the other X chromosome and the surrounding autosomes. (c) Indirect immunofluorescence showing the distribution of H4Ac12 on metaphase chromosomes (green, FITC). The location of the X chromosomes is indicated with the white arrows and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes lacks H4Ac12 signal, indicative of the Xi. This is the same X shown in b to be deficient for H2A-Bbd. (d) Merge of the staining patterns of H2A-Bbd and H4Ac12. The orange color indicates a direct overlap of the H2A-Bbd and H4Ac12 signals.
Mentions: Metaphase spreads were prepared from H2A-Bbd–transfected 293 cells and coimmunostained for regions of H4 acetylation. The Xi has previously been shown to be hypoacetylated at H4 at metaphase (Jeppesen and Turner 1993), indicating acetylation status as a reliable marker for the Xi. While one X chromosome (the cytologically identifiable Xa) showed staining equivalent to the autosomes, additional X chromosomes in each spread lacked any H4Ac12 signal, identifying them as Xi's. As evaluated in >120 metaphase spreads, the same X chromosomes were also deficient for H2A-Bbd staining (Fig. 6). In no case was H2A-Bbd found to stain the underacetylated Xi chromosome. An overlay of both H2A-Bbd and H4Ac12 shows an identical pattern of distribution at metaphase (Fig. 6 d).

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