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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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Indirect immunofluorescence of the H2A-Bbd banding pattern on the inactive X chromosome. Partial metaphase spreads of H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite rhodamine signal (orange) (a and a′) or Cy5 signal (pink) (b, b′, c, and c′). In all panels, the X chromosome is indicated with a white arrow. Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes was detected with FITC (green, a′) or rhodamine (red, b′ and c′).
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Figure 4: Indirect immunofluorescence of the H2A-Bbd banding pattern on the inactive X chromosome. Partial metaphase spreads of H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite rhodamine signal (orange) (a and a′) or Cy5 signal (pink) (b, b′, c, and c′). In all panels, the X chromosome is indicated with a white arrow. Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes was detected with FITC (green, a′) or rhodamine (red, b′ and c′).

Mentions: In some metaphase spreads from female cells, close examination of the weakly staining X chromosome by H2A-Bbd revealed a slight banding pattern on the Xi (Fig. 4). The staining was most prominent on several regions of the short arm of the chromosome and in the pericentromeric region, with occasional staining on the proximal long arm.


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)

Indirect immunofluorescence of the H2A-Bbd banding pattern on the inactive X chromosome. Partial metaphase spreads of H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite rhodamine signal (orange) (a and a′) or Cy5 signal (pink) (b, b′, c, and c′). In all panels, the X chromosome is indicated with a white arrow. Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes was detected with FITC (green, a′) or rhodamine (red, b′ and c′).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199617&req=5

Figure 4: Indirect immunofluorescence of the H2A-Bbd banding pattern on the inactive X chromosome. Partial metaphase spreads of H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite rhodamine signal (orange) (a and a′) or Cy5 signal (pink) (b, b′, c, and c′). In all panels, the X chromosome is indicated with a white arrow. Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes was detected with FITC (green, a′) or rhodamine (red, b′ and c′).
Mentions: In some metaphase spreads from female cells, close examination of the weakly staining X chromosome by H2A-Bbd revealed a slight banding pattern on the Xi (Fig. 4). The staining was most prominent on several regions of the short arm of the chromosome and in the pericentromeric region, with occasional staining on the proximal long arm.

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