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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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Expression and sequence analysis of a novel histone H2A variant, H2A-Bbd. (a) Human adult tissue Northern showing hybridization signal in testis using a probe derived from the coding sequence of the novel histone H2A variant. (b) PCR products of reverse transcribed poly(A)+ RNA. (1) marker; (2) water control; (3) female primary fibroblast cDNA; (4) female primary fibroblast, no reverse transcriptase control; (5) 293 cDNA; (6) 293, no reverse transcriptase control; (7) female lymphoblast cDNA; (8) female lymphoblast, no reverse transcriptase control. (c) Sequence of the novel histone H2A variant (782067), the H2A region of macroH2A (AAC39908), and three members of the human H2A family: H2A.1, a replication-linked H2A (CAB06031), the histone variant H2A.X (P16104), and the histone variant H2A.Z (P17317). The location of residues modified by acetylation (Ac) and ubiquitination (Ub) are indicated. The three α-helices (I, II, and III) of the histone fold domain are indicated. Alignments were made using GeneWorks® release 2.2.1 (IntelliGenetics).
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Figure 1: Expression and sequence analysis of a novel histone H2A variant, H2A-Bbd. (a) Human adult tissue Northern showing hybridization signal in testis using a probe derived from the coding sequence of the novel histone H2A variant. (b) PCR products of reverse transcribed poly(A)+ RNA. (1) marker; (2) water control; (3) female primary fibroblast cDNA; (4) female primary fibroblast, no reverse transcriptase control; (5) 293 cDNA; (6) 293, no reverse transcriptase control; (7) female lymphoblast cDNA; (8) female lymphoblast, no reverse transcriptase control. (c) Sequence of the novel histone H2A variant (782067), the H2A region of macroH2A (AAC39908), and three members of the human H2A family: H2A.1, a replication-linked H2A (CAB06031), the histone variant H2A.X (P16104), and the histone variant H2A.Z (P17317). The location of residues modified by acetylation (Ac) and ubiquitination (Ub) are indicated. The three α-helices (I, II, and III) of the histone fold domain are indicated. Alignments were made using GeneWorks® release 2.2.1 (IntelliGenetics).

Mentions: Using the nucleotide sequence of members of the human histone H2A family, we searched the public databases and identified a group of overlapping ESTs with distant homology to H2A that matched a predicted gene present in three intronless copies in Xq28 (Naylor et al. 1995). Two representative clones were obtained for the group and completely sequenced (AF254576). An open reading frame was identified from nucleotides 9–356, with the ATG at nucleotide 9 having a good match to the translation initiation start site consensus for vertebrates (CCCAGCAUGC versus GCCA/GCCAUGG) (Kozak 1991). To demonstrate expression of the gene, the coding sequence was used to probe a Northern blot, which identified a signal of ∼900 bp in testis (Fig. 1 a). The presence of the mRNA in a variety of cell lines and tissues was demonstrated by reverse transcription PCR (Fig. 1 b). Unlike conventional histone H2A genes (Dominski and Marzluff 1999), the cDNA sequence contained a polyA tail and has a consensus polyadenylation sequence of AATAAA. In addition, no vertebrate Histone Downstream Element consensus sequence of AAAGAG is obvious in the 3′ untranslated region (Williams and Marzluff 1995), nor is there a match to the consensus stem–loop sequence, suggesting that polyadenylation is the only form of 3′-end processing for this transcript. The presence of the polyA tail was confirmed by 3′RACE using a primer specific to the polyA start site in the ESTs in the database.


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)

Expression and sequence analysis of a novel histone H2A variant, H2A-Bbd. (a) Human adult tissue Northern showing hybridization signal in testis using a probe derived from the coding sequence of the novel histone H2A variant. (b) PCR products of reverse transcribed poly(A)+ RNA. (1) marker; (2) water control; (3) female primary fibroblast cDNA; (4) female primary fibroblast, no reverse transcriptase control; (5) 293 cDNA; (6) 293, no reverse transcriptase control; (7) female lymphoblast cDNA; (8) female lymphoblast, no reverse transcriptase control. (c) Sequence of the novel histone H2A variant (782067), the H2A region of macroH2A (AAC39908), and three members of the human H2A family: H2A.1, a replication-linked H2A (CAB06031), the histone variant H2A.X (P16104), and the histone variant H2A.Z (P17317). The location of residues modified by acetylation (Ac) and ubiquitination (Ub) are indicated. The three α-helices (I, II, and III) of the histone fold domain are indicated. Alignments were made using GeneWorks® release 2.2.1 (IntelliGenetics).
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Related In: Results  -  Collection

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Figure 1: Expression and sequence analysis of a novel histone H2A variant, H2A-Bbd. (a) Human adult tissue Northern showing hybridization signal in testis using a probe derived from the coding sequence of the novel histone H2A variant. (b) PCR products of reverse transcribed poly(A)+ RNA. (1) marker; (2) water control; (3) female primary fibroblast cDNA; (4) female primary fibroblast, no reverse transcriptase control; (5) 293 cDNA; (6) 293, no reverse transcriptase control; (7) female lymphoblast cDNA; (8) female lymphoblast, no reverse transcriptase control. (c) Sequence of the novel histone H2A variant (782067), the H2A region of macroH2A (AAC39908), and three members of the human H2A family: H2A.1, a replication-linked H2A (CAB06031), the histone variant H2A.X (P16104), and the histone variant H2A.Z (P17317). The location of residues modified by acetylation (Ac) and ubiquitination (Ub) are indicated. The three α-helices (I, II, and III) of the histone fold domain are indicated. Alignments were made using GeneWorks® release 2.2.1 (IntelliGenetics).
Mentions: Using the nucleotide sequence of members of the human histone H2A family, we searched the public databases and identified a group of overlapping ESTs with distant homology to H2A that matched a predicted gene present in three intronless copies in Xq28 (Naylor et al. 1995). Two representative clones were obtained for the group and completely sequenced (AF254576). An open reading frame was identified from nucleotides 9–356, with the ATG at nucleotide 9 having a good match to the translation initiation start site consensus for vertebrates (CCCAGCAUGC versus GCCA/GCCAUGG) (Kozak 1991). To demonstrate expression of the gene, the coding sequence was used to probe a Northern blot, which identified a signal of ∼900 bp in testis (Fig. 1 a). The presence of the mRNA in a variety of cell lines and tissues was demonstrated by reverse transcription PCR (Fig. 1 b). Unlike conventional histone H2A genes (Dominski and Marzluff 1999), the cDNA sequence contained a polyA tail and has a consensus polyadenylation sequence of AATAAA. In addition, no vertebrate Histone Downstream Element consensus sequence of AAAGAG is obvious in the 3′ untranslated region (Williams and Marzluff 1995), nor is there a match to the consensus stem–loop sequence, suggesting that polyadenylation is the only form of 3′-end processing for this transcript. The presence of the polyA tail was confirmed by 3′RACE using a primer specific to the polyA start site in the ESTs in the database.

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