Monoubiquitylation of H2A.Z distinguishes its association with euchromatin or facultative heterochromatin

Abstract

H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.

FIG. 1.

The H2A.Z-L1 and H2A.Z-C antibodies are specific for H2A.Z. (A) Amino acid sequence alignment of mammalian H2A and H2A.Z. The two most divergent regions, to which the H2A.Z antibodies were raised, are underlined in green. (B) An shRNA construct that targets H2A.Z mRNA is cotransfected with a construct that expresses H2B-GFP into 293T cells. Transfected (green fluorescing) and nontransfected (nongreen) cells were examined by immunofluorescence using the H2A.Z-L1 and H2A.Z-C antibodies. Both antibodies stain the untransfected but not the transfected cells, indicating that both antibodies recognize the endogenous H2A.Z but not H2A.

FIG. 2.

H2A.Z is excluded from pericentric heterochromatin in differentiated mouse fibroblasts. (A) Mouse 10T1/2 fibroblasts were stained using antibodies against H2A.Z and HP1α and were examined by immunofluorescence microscopy. Both H2A.Z antibodies show punctate nuclear staining but are excluded from the DAPI-dense and HP1α-enriched pericentric heterochromatin. (B) Metaphase chromosome spreads from mouse 10T1/2 cells show the exclusion of H2A.Z from the pericentric heterochromatin. The mutually exclusive localization of H2A.Z and HP1α is seen both at the pericentric heterochromatin and on the chromosome arms.

FIG. 3.

H2A.Z-containing mononucleosomes have higher levels of di-MeK4/tri-MeK4 H3 and lower levels of tri-MeK9 H3 than H2A-containing nucleosomes. (A) Representative agarose gel analysis showing complete MNase digestion of chromatin into single nucleosomes before being used in the immunoprecipitation procedure. The image is a negative image of the original ethidium bromide-stained gel, so the DNA band is black and the gel background is white. (B) 293T cells were either mock transfected, transfected with Flag-H2A, or transfected with Avi-H2A.Z, and total cell lysates from the two transfected cell samples were mixed before being used in the Flag immunoprecipitation assay. Flag-H2A, but not Avi-H2A.Z, was brought down by the M2 beads, indicating that the immunoprecipitation procedure is highly specific. (C) Flag-H2A.Z- or Flag-H2A-containing mononucleosomes are immunoprecipitated using the Flag antibody-coupled M2 beads. The amounts of immunoprecipitated H3 were normalized relative to each other, and the relative levels of various H3 modifications were examined using the indicated antibodies. (D) The same mononucleosome immunoprecipitation was repeated using C-terminally Flag-tagged H2A and H2A.Z. For both experiments (C and D), H2A.Z-containing nucleosomes are enriched for K4-methylated H3, whereas H2A-containing nucleosomes are enriched for K9-methylated H3. WB, Western blot; IP, immunoprecipitation.

FIG. 4.

Endogenous H2A.Z and transfected H2A.Z are monoubiquitylated in vivo. (A) 293T cells were transfected with constructs that express GFP, H3-GFP, wild-type (WT) H2A.Z-GFP, or mutant (KKRR) H2A.Z-GFP, in which K120 and 121 were mutated to arginines. For the samples shown at the center and right panels, the HA-ubiquitin (HA-Ub) expression construct was cotransfected with the GFP or histone-GFP construct. Western blotting with the GFP antibody shows that a fraction of H2A.Z-GFP migrates slower than the bulk of H2A.Z-GFP (marked by asterisks). Blotting with the HA antibody indicates that this slower-migrating band represents the ubiquitylated form of H2A.Z-GFP (uH2A.Z-GFP). In contrast, neither H3-GFP nor the mutant H2A.Z-GFP is monoubiquitylated. (B) Mapping of the sites of monoubiquitylation by mutagenesis. Expression constructs for the wild type or K-to-R mutants of H2A.Z-GFP were transfected into 293T cells and detected by Western blotting using a GFP antibody. Ponceau-stained histones were used as loading controls. (C) Transfection studies using various point mutants of N-terminally tagged H2A.Z show that K120, K121, and K125 all can be used as monoubiquitylation sites in vivo. In contrast, only K118 and K119 of H2A are sites of monoubiquitylation. (D) Total cell extracts were harvested from asynchronously growing (As) or mitotically arrested (M) human HT1080 or mouse C127 cells, and the endogenous histone levels were examined by Western blot analyses. While the H2A.Z-L1 antibody detected both the unmodified H2A.Z and uH2A.Z, the H2A.Z-C antibody detected only the unmodified H2A.Z. Total H3 was used as a loading control, and the increased levels of phosphorylated H3 were used as an indication of cells arrested at mitosis. (E) 293T cells were transfected with the vector control or plasmids that express wild-type H2A.Z-GFP, the K-to-R mutant of H2A.Z-GFP, or wild-type H2A-GFP, and whole-cell extracts were examined by Western blotting using H2A.Z-L1, H2A.Z-C, or GFP antibody.

FIG. 5.

uH2A.Z is enriched on the inactive X chromosome in human cells. (A and B) Metaphase chromosomes were harvested from human female IMR90 cells and cytospun onto glass slides, and the indicated antibodies (H2A.Z-L1, H2A.Z-C, and tri-MeK27) were used for immunofluorescence staining of the chromosome spreads. The inactive X chromosomes from spreads (identified by enrichment of tri-MeK27 levels) were enlarged in the inset at the top left or right corners. (C) 293T cells were transfected with an HA-ubiquitin (HA-Ub) expression plasmid, and metaphase chromosomes were harvested and analyzed by immunofluorescence staining using the indicated antibodies. One of the inactive X chromosomes from the 293T cells is enlarged in the inset at the top right corner. (D) A representative inactive X chromosome from IMR90 cells costained with the H2A.Z-C and a monoclonal antibody that recognizes di- and tri-MeK4 H3.

FIG. 6.

Antibodies directed against the C terminus of H2A.Z do not recognize the monoubiquitylated form of H2A.Z. Total cell lysates from 293T cells were examined by Western blot analyses using the indicated H2A.Z antibodies. Both the H2A.Z-C antibody and the commercially available antibody have difficulty detecting the endogenous monoubiquitylated H2A.Z. The bands marked by an asterisk represent nonspecific proteins within the total cell extracts that are recognized by the Abcam H2A.Z antibody.

FIG. 7.

Ubiquitylation of H2A.Z is not required for its incorporation onto the inactive X chromosomes. 293T cells were transfected with expression constructs for wild-type Flag-H2A (A), wild-type Flag-H2A.Z (B), or the nonubiquitylatable mutant (K3R3) form of Flag-H2A.Z (C). Metaphase chromosomes were isolated from the transfected cells and were immunostained using the Flag and tri-MeK27 H3 antibodies. The chromosomes enriched for tri-meK27 H3 (marked by white arrowheads) represent the inactive X chromosomes. Since the panels show only part of the chromosome spreads, not all of the inactive X chromosomes present per cell are shown.

FIG. 8.

Expression of shRNA targeting RING1b reduces the levels of ubiquitylated H2A.Z. (A) 293T cells were transfected with the control scrambled shRNA construct (Ctrl shRNA) or with the shRNA construct targeting human RING1b along with either the empty vectors (untransfected) or the Flag-H2A or Flag-H2A.Z construct. The total cell lysate was harvested, and the expression of Flag-H2A and Flag-H2A.Z was detected by Western blotting using the Flag-M2 antibody. As shown, the RING1B shRNA, but not the control shRNA, specifically knocked down expression of the endogenous RING1b protein as well as the monoubiquitylated forms of Flag-H2A and Flag-H2A.Z. The Western blot using the H3 antibody is shown as a loading control. (B) 293T cells were transfected with the control or the RING1b shRNA constructs, along with empty vectors or constructs expressing Bmi1 and Flag-RING1b. The effects of these constructs on the ubiquitylation levels of endogenous H2A.Z were examined by Western blotting using the H2A.Z-L1 antibody. Note that the lysate from cells transfected with the Flag-RING1b and Bmi1 contains both the endogenous and overexpressed Flag-tagged RING1b, and these two versions of RING1b were not well resolved on the gel due to the high percentage (15%) of acrylamide used.