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. 2006 Jul;26(14):5325-35.
doi: 10.1128/MCB.00584-06.

Analysis of human histone H2AZ deposition in vivo argues against its direct role in epigenetic templating mechanisms

Antoine Viens  1 , Undine Mechold, Franck Brouillard, Cristele Gilbert, Philippe Leclerc, Vasily Ogryzko
Affiliations

Analysis of human histone H2AZ deposition in vivo argues against its direct role in epigenetic templating mechanisms

Antoine Viens et al. Mol Cell Biol. 2006 Jul.

Abstract

Chromatin is considered to be a principal carrier of epigenetic information due to the ability of alternative chromatin states to persist through generations of cell divisions and to spread on DNA. Replacement histone variants are novel candidates for epigenetic marking of chromatin. We developed a novel approach to analyze the chromatin environment of nucleosomes containing a particular replacement histone. We applied it to human H2AZ, one of the most studied alternative histones. We find that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark.

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Figures

FIG. 1.
FIG. 1.
Separate isolation of H2AZ-containing histone octamer and its nucleosome neighbors. (A) epi:H2A and epi:H2AZ are incorporated into chromatin. Mitotic cells expressing epi:H2A (top) and epi:H2AZ (bottom) were stained with anti-HA antibody (left) and Hoechst (right). (B) Expression levels of epi:H2AZ. Left panel: Western analysis of histones from control cells (ctrl) and from cells expressing C-terminally tagged H2AZ (ZC) and N-terminally tagged H2AZ (ZN) with antibody against H2AZ. Right panel: the same samples analyzed with anti-HA antibody. Numbers to the left of the panels indicate molecular mass in kDa. (C) The elution scheme. After immobilization of epi:H2AZ-containing chromatin on the Ni2+-agarose, the DNA together with the histone octamers from the neighboring nucleosomes was evaluated by 2 M NaCl. Subsequently, the histone octamer containing the epitope-tagged histone was eluted with 250 mM imidazole. (D) Analysis of the NaCl and imidazole fractions. NaCl (left) and imidazole (right) fractions were analyzed for protein (top) and DNA (bottom) content. Positions of regular and epitope-tagged histones are indicated to the left of the top panel. Positions of the tri-, di-, and mononucleosomal (mono) DNA are indicated on the left of the bottom panel. EtBr, ethidium bromide.
FIG. 2.
FIG. 2.
Association of H2AZ with markers of active and repressed chromatin. (A) Staining of C2C12 cells with the antibodies against trimethylated histones. C2C12 cells were stained with antibodies specific for trimethylated lysines H3K4, H4K20, H3K9, and H3K27 (right) and with Hoechst (left). (B) Western analysis of H2AZ- and H2A-containing nucleosomes with the same antibodies. Histones H3 and H4 from the epi:H2A (right of each panel; labeled “A”)- and epi:H2AZ (left; “Z”)-containing octamers were analyzed by Western analysis with antibodies specific for methylated lysines H3K4, H3K9, H3K27, and H4K20. Octamers obtained by both N-terminal (left panel) and C-terminal (right panel) tagging were analyzed. The bottom three lanes correspond to antibodies against H4, H3 histones, and HA epitope used for normalization. (C) H2AZ nucleosome is enriched by cytosine methylation. A slot blot analysis of a serial twofold dilution of DNA from the nucleosome preparations shown in Fig. 1D, bottom, is shown. After fixation on filters, the presence of methylated cytosine in DNA was detected with anti-methylcytosine antibody. Shown are the concentrations of DNA at the start and the end of serial dilutions.
FIG. 3.
FIG. 3.
Analysis of colocalization of H2A and H2AZ. NIH 3T3 cells were transfected with vectors expressing CFP-H2A and YFP-H2A fusions (left) or CFP-H2A and YFP-H2AZ fusions (right). The top shows typical images of transfected cells, with green and red pseudocoloring of the CFP and YFP signals, respectively. Shown on the bottom are the results of typical colocalization analysis. The color coding indicates the frequencies (with red corresponding to the highest and blue corresponding to the lowest) of the occurrence of pixels with given intensities of CFP (CA) and YFP (YA, YZ) signals. The correlation coefficients with the deviations obtained by averaging the colocalization coefficients measured for 10 different cells are shown on the left top corner of each diagram.
FIG. 4.
FIG. 4.
Analysis of the endogenous H2A bands in the H2AZ-containing octamer and the neighboring nucleosomes. (A) Western analysis results. Histones from the epi:H2AZ- and epi:H2A-containing octamers or their neighborhoods (oZ, oA, nZ, and nA, respectively), were analyzed with anti-HA (top), anti-H2AZ (middle), and anti-H4 (bottom) antibodies. The lower part of the anti-H2AZ blot, overexposed to reveal endogenous H2AZ, is also shown (αH2AZ*). (B) Results of a mass spectrometry analysis of the H2A(H2AZ) bands in identification mode. The bands corresponding to the H2A(H2AZ) histone from different samples (shown boxed on the left panel) were excised and analyzed by nano-LC/MS-MS in identification mode. Top left panel: A, e:H2A octamer and neighborhood; Z, e:H2AZ octamer and neighborhood. The positions and intensities of the peaks corresponding to the H2A- and H2AZ-specific peptides are shown on the chromatograms (right panel and bottom). (C) Alignment of the H2A and H2AZ peptide sequences. The locations of all peptides used for identification of the endogenous H2A bands shown in panel B are boxed. Indicated are the A/Z and the Z peptides used for quantification of the H2AZ content in the endogenous H2A/Z bands shown in Fig. 5. (D) Confirmation of peak identity by MS-MS analysis. Left: MS2 spectra of the common A/Z peptide AGLQFPVGR from the epi:H2AZ band. Right: MS2 spectra of the specific Z peptide GDEELDSLIK from the epi:H2AZ band. Shown to the left of each panel are the expected ions from the Y and B series. Shown to the right are the detected ions from the Y and B series. AA, amino acid.
FIG. 5.
FIG. 5.
No enrichment by the second H2AZ molecule in the H2AZ-containing octamer or in its neighborhood. (A) Analysis of the chromatin containing C-terminally tagged H2AZ. The bands corresponding to the untagged H2A(H2AZ) histone from different samples (SDS-PAGE gel shown boxed on the top left panel) were excised and analyzed by nano-LC/MS-MS set to detect the H2AZ-specific (GDEELDSLIK) and common (AGLQFPVGR) peptides (right panel; Z and A/Z, respectively). The ratios of the intensities of the A/Z and Z peaks are shown on the top right corner of each chromatogram. Top left panel: O, octamer; N, neighborhood; A, epi:H2A octamer and neighborhood; Z, epi:H2AZ octamer and neighborhood; Total HeLa, histones obtained from HeLa cells by acid extraction. (B) Analysis of the chromatin containing N-terminally tagged H2AZ and various controls. Similar analyses were performed on the chromatin with N-terminally tagged H2AZ and in various control experiments. Octamer, untagged H2A(H2AZ) band from the epi:H2AZ-containing octamer; Mono, untagged H2A(H2AZ) band from the epi:H2AZ-containing mononucleosome (see panel C); e:H2AZ, epitope-tagged H2AZ band from the epi:H2AZ-containing octamer; IP αH2AZ, untagged H2A(H2AZ) band from the chromatin fragments obtained by immunoprecipitation with antibody against native H2AZ. The identities of the H2AZ-specific peaks were confirmed by MS-MS spectra shown on the right and at the bottom. (C) Purification of epi:H2AZ-containing mononucleosome. Left: analysis of DNA from the mononucleosome preparation. M1 and M2, DNA ladders. In lane MN, the DNA band from the mononucleosome preparation of approximately 150 bp is indicated by an arrow. Right: SDS-PAGE analysis of histone composition of the mononucleosome. M, Seeblue marker (Invitrogen); MN, bands corresponding to histones H3, H4, H2A(H2AZ), H2B, and epi:H2AZ. The boxed protein band corresponding to untagged H2A(H2AZ) histone was excised and analyzed by use of LC/MS-MS (panel B, Mono). (D) Analysis of posttranslational modifications of the nucleosome neighbors of the epi:H2AZ- and epi:H2A-containing nucleosomes. Histones H3 and H4 from the C-terminally tagged epi:H2A (right of each panel; “A” column) and epi:H2AZ (left; “Z”) nucleosome neighborhoods were analyzed by Western analysis with antibodies specific for methylated lysines H3K4, H3K9, H3K27, and H4K20. The two bottom panels correspond to antibodies against H4 and H3 histones used as normalization controls.
FIG. 6.
FIG. 6.
Model of a replacement histone action in epigenetic templating. A replacement histone in the chromatin could act as a self-perpetuating epigenetic mark, signaling for recruitment of its own deposition machinery. We consider the consequences of the histone loss in three cases: cases I and II have another molecule of the same histone present either in the close neighborhood (case I) or in the same octamer (case II), and case III has no second molecule present. In both cases I and II, the second molecule of the replacement histone provides a memory that allows cells to recover the original state of chromatin by recruiting the deposition machinery to the site. In the third case, the information is lost and the recovery of the original state is not possible.
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