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

Chromatin on the mammalian inactive X chromosome differs in a number of way s from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactiv e X chromosome in female cells. Here, we report the identification and loca lization of a novel and more distant histone variant, designated H2A-Bbd, t hat is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protei n-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 a nd on metaphase chromosomes. Chromatin fractionation demonstrates associati on of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease -digested chromatin by sucrose gradient ultracentrifugation shows cofractio nation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is inc orporated into nucleosomes as a substitute for the core histone H2A, This f inding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with tr anscriptionally active regions of the genome. The distribution of H2A-Bbd t hus distinguishes chromatin on the active and inactive X chromosomes.