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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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Association of H2A-Bbd with acid-extracted histone proteins in chromatin fractions and copurification with nucleosomes by sucrose gradient ultracentrifugation. (a) Coomassie stain of an 18% polyacrylamide gel of chromatin fractions. (1) Proteins from the chromatin pellet fraction from 293 cells. (2) Proteins extracted from the 293 chromatin pellet under acidic conditions. (3) Proteins from the chromatin pellet fraction from a stable H2A-Bbd–transfected 293 cell line. (4) Proteins extracted from the stable H2A-Bbd–transfected 293 chromatin pellet under acidic conditions. Sizes are given in kilodaltons. The location of histone H1 and the core histones (H2A, H2B, H3, and H4) are indicated. (b) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope-tagged H2A-Bbd in the acid-extracted fraction of the H2A-Bbd–stable transfected 293 cell line only (4). (c) Coomassie stain of an 18% polyacrylamide gel of nucleosome containing sucrose gradient fractions. (5) Nucleosomes from a nontransfected 293 cell line. (6) Nucleosomes from a stable H2A-Bbd–transfected 293 cell line. (d) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope tagged H2A-Bbd in the nucleosomal fraction of the H2A-Bbd stable transfected 293 cell line only (6).
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Figure 7: Association of H2A-Bbd with acid-extracted histone proteins in chromatin fractions and copurification with nucleosomes by sucrose gradient ultracentrifugation. (a) Coomassie stain of an 18% polyacrylamide gel of chromatin fractions. (1) Proteins from the chromatin pellet fraction from 293 cells. (2) Proteins extracted from the 293 chromatin pellet under acidic conditions. (3) Proteins from the chromatin pellet fraction from a stable H2A-Bbd–transfected 293 cell line. (4) Proteins extracted from the stable H2A-Bbd–transfected 293 chromatin pellet under acidic conditions. Sizes are given in kilodaltons. The location of histone H1 and the core histones (H2A, H2B, H3, and H4) are indicated. (b) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope-tagged H2A-Bbd in the acid-extracted fraction of the H2A-Bbd–stable transfected 293 cell line only (4). (c) Coomassie stain of an 18% polyacrylamide gel of nucleosome containing sucrose gradient fractions. (5) Nucleosomes from a nontransfected 293 cell line. (6) Nucleosomes from a stable H2A-Bbd–transfected 293 cell line. (d) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope tagged H2A-Bbd in the nucleosomal fraction of the H2A-Bbd stable transfected 293 cell line only (6).

Mentions: To address the question of association of H2A-Bbd with core histones in the nucleosome, the myc epitope-tagged version of the protein was transfected into 293 cells, and a stable cell line established. Chromatin was isolated from the cells, and histones were extracted under acidic conditions and separated from the remaining chromatin by centrifugation. By Western analysis, H2A-Bbd was found to cofractionate with histones, indicating that H2A-Bbd behaves like core histones (Fig. 7, a and b). The same result was obtained using an anti–myc mAb (data not shown). The chromatin fractions were tested for the presence of a number of nonhistone chromatin proteins in the same way, and none was found to cofractionate with the histones (data not shown).


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)

Association of H2A-Bbd with acid-extracted histone proteins in chromatin fractions and copurification with nucleosomes by sucrose gradient ultracentrifugation. (a) Coomassie stain of an 18% polyacrylamide gel of chromatin fractions. (1) Proteins from the chromatin pellet fraction from 293 cells. (2) Proteins extracted from the 293 chromatin pellet under acidic conditions. (3) Proteins from the chromatin pellet fraction from a stable H2A-Bbd–transfected 293 cell line. (4) Proteins extracted from the stable H2A-Bbd–transfected 293 chromatin pellet under acidic conditions. Sizes are given in kilodaltons. The location of histone H1 and the core histones (H2A, H2B, H3, and H4) are indicated. (b) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope-tagged H2A-Bbd in the acid-extracted fraction of the H2A-Bbd–stable transfected 293 cell line only (4). (c) Coomassie stain of an 18% polyacrylamide gel of nucleosome containing sucrose gradient fractions. (5) Nucleosomes from a nontransfected 293 cell line. (6) Nucleosomes from a stable H2A-Bbd–transfected 293 cell line. (d) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope tagged H2A-Bbd in the nucleosomal fraction of the H2A-Bbd stable transfected 293 cell line only (6).
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Figure 7: Association of H2A-Bbd with acid-extracted histone proteins in chromatin fractions and copurification with nucleosomes by sucrose gradient ultracentrifugation. (a) Coomassie stain of an 18% polyacrylamide gel of chromatin fractions. (1) Proteins from the chromatin pellet fraction from 293 cells. (2) Proteins extracted from the 293 chromatin pellet under acidic conditions. (3) Proteins from the chromatin pellet fraction from a stable H2A-Bbd–transfected 293 cell line. (4) Proteins extracted from the stable H2A-Bbd–transfected 293 chromatin pellet under acidic conditions. Sizes are given in kilodaltons. The location of histone H1 and the core histones (H2A, H2B, H3, and H4) are indicated. (b) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope-tagged H2A-Bbd in the acid-extracted fraction of the H2A-Bbd–stable transfected 293 cell line only (4). (c) Coomassie stain of an 18% polyacrylamide gel of nucleosome containing sucrose gradient fractions. (5) Nucleosomes from a nontransfected 293 cell line. (6) Nucleosomes from a stable H2A-Bbd–transfected 293 cell line. (d) Immunoblot analysis of chromatin fractions from 293 and a stable H2A-Bbd–transfected 293 cell line. A clear 17-kD signal can be seen for the epitope tagged H2A-Bbd in the nucleosomal fraction of the H2A-Bbd stable transfected 293 cell line only (6).
Mentions: To address the question of association of H2A-Bbd with core histones in the nucleosome, the myc epitope-tagged version of the protein was transfected into 293 cells, and a stable cell line established. Chromatin was isolated from the cells, and histones were extracted under acidic conditions and separated from the remaining chromatin by centrifugation. By Western analysis, H2A-Bbd was found to cofractionate with histones, indicating that H2A-Bbd behaves like core histones (Fig. 7, a and b). The same result was obtained using an anti–myc mAb (data not shown). The chromatin fractions were tested for the presence of a number of nonhistone chromatin proteins in the same way, and none was found to cofractionate with the histones (data not shown).

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