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

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.

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).

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).

Figure 2

Nuclear distribution of H2A-Bbd at interphase in primary female fibroblast cells showing the zone of exclusion around the Barr body and X chromosome, and the distribution of macroH2A in 46,XY and 47,XXX primary fibroblast cell nuclei. (a) Transfected female cell showing the nuclear distribution of a COOH-terminal GFP-tagged H2A-Bbd by indirect immunofluorescence (green). The region of exclusion is indicated with the white arrow. (a′) DAPI staining of the same nucleus reveals the condensed Xi in the form of the Barr body at the periphery of the nucleus (white arrow). (b) Indirect immunofluorescence of a transfected female cell showing the nuclear distribution of a COOH-terminal myc-tagged H2A-Bbd (red, TR), stained with a anti–myc mAb followed by goat anti–mouse IgG conjugated with Texas red. The exclusion is indicated with a white arrow. (b′) The DAPI image of the same nucleus indicates the position of the Barr body. (c) Female cell transfected with myc-tagged H2A-Bbd showing the nuclear distribution of H2A-Bbd by indirect immunofluorescence (green, FITC) merged with the FISH signals for a human X alpha satellite probe (orange, rhodamine). One of the two X centromere signals is located within a deficient region and is indicated with a white arrow. (c′) The DAPI image of the same nucleus indicates the location of the X alpha satellite FISH probe that is contained within the H2A-Bbd exclusion zone. (d) Transfected female cell showing the even nuclear distribution of a COOH-terminal myc-epitope–tagged H2B construct by indirect immunofluorescence (green, FITC) superimposed with two X alpha satellite FISH signals (orange, rhodamine). Neither X chromosome is located within a region deficient in H2B staining. (d′) DAPI stain of the same nucleus is shown. (e) Transfected interphase male cell showing the nuclear distribution of a COOH-terminal myc-epitope–tagged macroH2A construct by indirect immunofluorescence (green, FITC). (e′) The location of the single X chromosome is shown merged with the DAPI stain of the same nucleus (orange, rhodamine). No MCBs characteristic of an Xi can be seen in the nucleus. (f) Indirect immunofluorescence of a female interphase triple X cell transfected with macroH2A showing the nuclear location of two MCBs (green, FITC) indicated by white arrows. (f′) The DAPI stain of the same nucleus merged with the X alpha satellite FISH (orange, rhodamine) shows two X signals to be coincident with the MCB indicative of Xi's. The third X chromosome is not associated with an MCB.

Figure 3

Chromosomal localization of H2A-Bbd on metaphase chromosomes from the female embryonic kidney carcinoma cell line 293. (a) Partial metaphase spread of a H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite cyanine 5.18 (Cy5) FISH signals (pink). Three X chromosomes are indicated with white arrows. (b) Indirect immunofluorescence of H2A-Bbd distribution on the same partial metaphase spread. Two of the X chromosomes do not stain with H2A-Bbd, while one (the cytologically marked Xa) shows a similar pattern to the surrounding autosomes (red, TR). The X alpha satellite Cy5 signal is shown (pink) and the positions of the X chromosomes are indicated with white arrows.

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′).

Figure 6

Female metaphase chromosomes from a H2A-Bbd–transfected 293 cell, counterstained for histone H4 acetylation at lysine-12. (a) DAPI stain of metaphase chromosomes merged with the FISH signal of an X alpha satellite probe (pink, Cy5). The positions of the two X chromosomes is indicated with white arrows. (b) Indirect immunofluorescence of the H2A-Bbd distribution along the metaphase chromosomes (red, rhodamine). The location of the two X chromosomes is shown with the white arrow, and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes is deficient for H2A-Bbd when compared with the other X chromosome and the surrounding autosomes. (c) Indirect immunofluorescence showing the distribution of H4Ac12 on metaphase chromosomes (green, FITC). The location of the X chromosomes is indicated with the white arrows and the centromere is marked by the FISH signal from X alpha satellite probe (pink, Cy5). One of the two X chromosomes lacks H4Ac12 signal, indicative of the Xi. This is the same X shown in b to be deficient for H2A-Bbd. (d) Merge of the staining patterns of H2A-Bbd and H4Ac12. The orange color indicates a direct overlap of the H2A-Bbd and H4Ac12 signals.

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.

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).