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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 primary fibroblast interphase cell showing the nuclear distribution of H2A-Bbd and histone H4 acetylation. (a) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution by indirect immunofluorescence (red, TR). The white arrow indicates the location of the Xi-associated exclusion. (b) Indirect immunofluorescence showing the distribution of acetylation of histone H4 at lysine-12 (green, FITC). A distinct region lacking acetylation is indicated with an arrow. (c) Merge of the H2A-Bbd and H4Ac12 staining patterns. The nucleus has an orange appearance due to complete overlap of the two distributions. The exclusion zone indicated with the white arrow is clearly underacetylated and deficient for H2A-Bbd.
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Figure 5: Female primary fibroblast interphase cell showing the nuclear distribution of H2A-Bbd and histone H4 acetylation. (a) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution by indirect immunofluorescence (red, TR). The white arrow indicates the location of the Xi-associated exclusion. (b) Indirect immunofluorescence showing the distribution of acetylation of histone H4 at lysine-12 (green, FITC). A distinct region lacking acetylation is indicated with an arrow. (c) Merge of the H2A-Bbd and H4Ac12 staining patterns. The nucleus has an orange appearance due to complete overlap of the two distributions. The exclusion zone indicated with the white arrow is clearly underacetylated and deficient for H2A-Bbd.

Mentions: Histone H4 is posttranslationally modified by acetylation at four lysine residues in the NH2-terminal tail of the protein (Luger and Richmond 1998; Wolffe and Hayes 1999; Spencer and Davie 1999). Euchromatic regions of the genome are enriched for acetylated H4 isoforms (O'Neill and Turner 1995), while heterochromatic domains (including the Xi) lack acetylation (Jeppesen and Turner 1993; O'Neill and Turner 1995; Boggs et al. 1996; Gilbert and Sharp 1999). Using antisera to H4Ac12, we compared the pattern of H4-acetylation to the distribution of H2A-Bbd. Both H2A-Bbd and acetylated H4 localize throughout the nucleus, with the exception of the exclusion zone corresponding to the Xi and the Barr body (Fig. 5). Overlaying the two distributions reveals almost identical patterns, indicating that H2A-Bbd is tightly associated with acetylated euchromatic regions of the genome.


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 primary fibroblast interphase cell showing the nuclear distribution of H2A-Bbd and histone H4 acetylation. (a) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution by indirect immunofluorescence (red, TR). The white arrow indicates the location of the Xi-associated exclusion. (b) Indirect immunofluorescence showing the distribution of acetylation of histone H4 at lysine-12 (green, FITC). A distinct region lacking acetylation is indicated with an arrow. (c) Merge of the H2A-Bbd and H4Ac12 staining patterns. The nucleus has an orange appearance due to complete overlap of the two distributions. The exclusion zone indicated with the white arrow is clearly underacetylated and deficient for H2A-Bbd.
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Related In: Results  -  Collection

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Figure 5: Female primary fibroblast interphase cell showing the nuclear distribution of H2A-Bbd and histone H4 acetylation. (a) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution by indirect immunofluorescence (red, TR). The white arrow indicates the location of the Xi-associated exclusion. (b) Indirect immunofluorescence showing the distribution of acetylation of histone H4 at lysine-12 (green, FITC). A distinct region lacking acetylation is indicated with an arrow. (c) Merge of the H2A-Bbd and H4Ac12 staining patterns. The nucleus has an orange appearance due to complete overlap of the two distributions. The exclusion zone indicated with the white arrow is clearly underacetylated and deficient for H2A-Bbd.
Mentions: Histone H4 is posttranslationally modified by acetylation at four lysine residues in the NH2-terminal tail of the protein (Luger and Richmond 1998; Wolffe and Hayes 1999; Spencer and Davie 1999). Euchromatic regions of the genome are enriched for acetylated H4 isoforms (O'Neill and Turner 1995), while heterochromatic domains (including the Xi) lack acetylation (Jeppesen and Turner 1993; O'Neill and Turner 1995; Boggs et al. 1996; Gilbert and Sharp 1999). Using antisera to H4Ac12, we compared the pattern of H4-acetylation to the distribution of H2A-Bbd. Both H2A-Bbd and acetylated H4 localize throughout the nucleus, with the exception of the exclusion zone corresponding to the Xi and the Barr body (Fig. 5). Overlaying the two distributions reveals almost identical patterns, indicating that H2A-Bbd is tightly associated with acetylated euchromatic regions of the genome.

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