H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization

Abstract

The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

Figure 1.

Identification of Z.2.2. (A) qPCR with cDNA from different human cell lines and tissues using primers specific for Z.2.1 and Z.2.2. Data were normalized to HPRT1 and HMBS expression levels. Controls generated without reverse transcriptase (no RT) were used to assess amplification threshold. Shown are the levels of Z.2.2 mRNA as percentages of total Z.2 transcripts (Z.2.1 + Z.2.2). For an evaluation of absolute expression levels see Supplementary Figure S1C and D. (B) Amino acid alignment of human Z.1, Z.2.1 and Z.2.2 proteins using ClustalW Alignment (MacVector 10.0.2). Identical amino acids are highlighted in dark gray, similar amino acids in light gray and changes are set apart on white background. Known acetylation sites are depicted with stars and ubiquitination sites with circles. A schematic representation of the secondary structure of Z.1 and Z.2.1 is shown below the alignment, including depiction of the H3/H4 docking domain (50). M6 and M7 boxes indicate regions important for H2A.Z-specific biological functions in D. melanogaster (60). (C) IB analyses of RP–HPLC purified fractions from different human (HEK293, HeLa, HeLa Kyoto and U2OS) and mouse (NIH3T3) cell lines using a polyclonal rabbit αZ.2.2 and αH2A.Z (αZ, C-terminal) antibodies. Recombinant Z.2.2 protein (rZ.2.2) was loaded in the first lane as positive control for αZ.2.2 antibody. Similar results were obtained when using a monoclonal rat αZ.2.2 antibody (data not shown).

Figure 2.

Z.2.2 localizes to the nucleus and is partially incorporated into chromatin. (A) Fluorescence imaging of stably transfected HeLa Kyoto cells shows nuclear localization of all GFP-H2A variants (middle). DNA was counterstained with DAPI (top). Overlay of both channels in color is shown at the bottom (Merge; GFP: green, DAPI: blue). Scale bar = 5 µm. (B) Deconvolved images of metaphase spreads of HeLa Kyoto cells stably expressing GFP-H2A variants (middle). Merged images in color are shown below (GFP: green; DAPI: blue). Scale bar = 10 µm. (C) Chromatin from HeLa Kyoto cells stably expressing GFP-Z.2.2 was digested with MNase followed by a purification of mononucleosomes using sucrose gradient centrifugation. Isolated DNA from subsequent sucrose gradient fractions was analyzed by agarose gel electrophoresis (left). Fractions containing pure mononucleosomes (marked with asterisk) were combined and analyzed by IB (right) using αGFP antibody for the presence of GFP-Z.2.2 (top), and αH3 (bottom).

Figure 3.

The majority of Z.2.2 protein is nuclear soluble and highly mobile in a sequence-dependent manner. (A) HK-GFP cells were subjected to biochemical fractionation. Fractions sol and chr of identical cell equivalents were probed in IB with αGFP (top), αH2A (middle) and αGAPDH (bottom). (B) FRAP quantification curves of average GFP signal relative to fluorescence intensity prior to bleaching are depicted for GFP, GFP-tagged wild-type H2A variants, linker histone H1.0 and heterochromatin protein 1α (HP1α). Mean curves of 10–29 cells are shown for each construct. Error bars are omitted for clarity. (C) FRAP quantification curves similar to (B) are depicted for GFP, GFP-tagged wild-type H2A, Z.2.1, Z.2.2 and mutant constructs. (D) Quantitative evaluation of FRAP curves. Plot shows calculated mobility fraction sizes of different wild-type and mutant H2A variant constructs, as well as H1.0 and HP1α, based on bi-exponential fitting of FRAP data. Error bars indicate SD (see Supplementary Figure S3 for long-term FRAP and for numerical values).

Figure 4.

Z.2.2 associates with H2A.Z-specific SRCAP and TIP60 chaperone complexes. GFP-pull-downs for H2A.Z-specific chaperone complexes are shown. HK cells stably expressing GFP-Z.2.1 (A) and GFP-Z.2.2 (B) were SILAC-labeled and subjected to single-step affinity purifications of soluble nuclear proteins in a ‘forward’ (GFP-Z.2.1) or ‘reverse’ (GFP-Z.2.2) pull-down using GFP nanotrap beads. In each panel the ratio of the identified proteins after MS is plotted. Proteins known to interact with H2A.Z are indicated in the following way: members of the SRCAP complex in red, members of the TIP60 complex in blue and shared subunits in purple. Potential novel H2A.Z-interacting proteins are shown as green dots (‘other outliers’) and are distinguished from background binders (gray dots) and contaminants (yellow dots). See also Supplementary Table S1 for a list of all identified proteins. (C) List of the SRCAP and TIP60 complex members and their normalized binding intensity to Z.2.1 or Z.2.2. Note that for comparison reasons the obtained H/L ratios of GFP-Z.2.2 binders (numbers in brackets) were calculated in the corresponding L/H ratios. See also Supplementary Table S1 for a list of all identified proteins and their normalized H/L ratios.

Figure 5.

Z.2.1 and Z.2.2 are actively deposited into chromatin and are under-represented at replication foci. C127 cells transiently expressing GFP-Z.2.1 (left) and -Z.2.2 (right) were pulse labeled with EdU to visualize replication foci and to identify S-phase stages. DNA was counterstained with DAPI and analyzed by wide-field deconvolution microscopy. To remove the unbound fraction in GFP-Z.2.2 expressing cells, an in situ extraction was performed prior to fixation. Cells in early, middle and late S-phases were distinguished due to their characteristic differential EdU replication labeling patterns of eu- and heterochromatic regions. Merged images in color are shown alongside (GFP: green; EdU: red; DAPI: blue). Scale bar = 5 µm.

Figure 6.

Z.2.2 does not constitute stable histone octamers with H2B, H3 and H4 in vitro. (A) Size exclusion chromatography of refolding reactions using recombinant human H3, H4 and H2B proteins together with either H2A (solid line) or Bbd (dashed line) (left overlay) or with either Z.2.1 (solid line) or Z.2.2 (dashed line) (right overlay). Peaks corresponding to aggregates, histone octamers, tetramers or dimers are labeled respectively. (B) Fractions corresponding to H2A-containing octamers, Bbd-containing tetramers and dimers (left) or Z.2.1-containing octamers and Z.2.2-containing tetramers and dimers (right) were analyzed by 18% SDS–PAGE and stained with Coomassie brilliant blue.

Figure 7.

Z.2.2-containing nucleosomes are less resistant to MNase digestion and increased ionic strength. (A) H2A, Z.2.1 or Z.2.2 containing nucleosomes were assembled on DNA by salt gradient deposition, incubated at 4°C or 37°C to evaluate DNA positioning and separated by a native 5% PAGE gel. (B) Agarose-gel-electro-eluted material from (A) was analyzed by 18% SDS–PAGE and Coomassie stained to evaluate stoichiometry of histones after nucleosome assembly (top). Stars indicate H2A variants that were used for assembly. Further evaluation of histone stoichiometry after nucleosome assembly was done by IB using a LI-COR instrument (bottom). Assembled nucleosomes containing Z.2.1 or Z.2.2 were immunoblotted and the amount of histones was visualized using an αH3 antibody (top) and an N-terminal αZ antibody (recognizes all H2A.Z variants, bottom). (C) Mononucleosomes containing either H2A, Z.2.1 or Z.2.2 were digested with increasing concentrations of MNase and extracted DNA was separated using Bioanalyzer. Stars indicate DNA length of 146 bp. For detailed electropherogram analyses of fragment lengths in each sample see Supplementary Figure S5. (D) Mononucleosomes containing either H2A, Z.2.1 or Z.2.2 histones together with double dye labeled DNA were incubated with increasing amounts of salt. smFRET measurement values of each salt concentration were normalized to 0 mM NaCl. Error bars represent SEM of six measurements. (E) Chromatin from HK-GFP cells was isolated and incubated with increasing amounts of salt. Chromatin-bound histones were precipitated and detected by IB using αGFP antibody. Staining with αH2A was used as loading control.

Figure 8.

Unique Z.2.2 C-terminal amino acids cause significant changes in protein and nucleosome structure. (A) In silico models of Z.1, Z.2.1¹¹³ and Z.2.2 C-terminal C-chains (yellow; from amino acids 84 to C-terminus, including the complete docking domain) together with the E-chain of histone H3 (blue; amino acids 33–60, including αN-helix). Side (left) and frontal views (right) of four MD simulations are shown respectively. See Supplementary Figure S7 for complete in silico model of H2A.Z-containing nucleosome. (B) Simulated distances between peptide backbone NH of amino acids 112 in H2A.Z (His or Cys, respectively) variants and the oxygen in the Gln55 sidechain in H3 based on in silico nucleosome models containing either Z.1 (white), Z.2.1¹¹³ (light gray) or Z.2.2 (dark gray) proteins. Error bars represent SD of four independent simulations. (C) Simulated mobility measuring B-factor values between amino acids 108 and 113 in respective H2A.Z variant C-termini. Error bars represent SD of four independent simulations. (D) Simulated interaction energy between tetramer versus respective H2A.Z variant-containing dimers.