While many studies have linked the function of H2A.Z to transcriptional regulation, its exact function in this process has remained enigmatic, since abundant evidence supports both positive and negative roles for this histone variant in transcription (reviewed in reference 14
). Much of our current understanding comes from studies of the H2A.Z homolog Htz1 of Saccharomyces cerevisiae
. Loss of Htz1 leads to defects in transcriptional activation of the PHO5 and GAL1 genes, as well as disruption of the transcriptional silencing of the HMR and telomeric loci of this organism (9
). Global gene expression profiling of Htz1 deletion mutants suggests that this variant functions to counteract the spreading of Sir2-mediated heterochromatin-type silencing (21
). Studies combining chromatin immunoprecipitation and microarray analysis indicate that Htz1 is specifically localized to the 5′ end of genes, and furthermore, Htz1 is preferentially associated with inactive genes (13
). Interestingly, deletion of Htz1 does not affect transcriptional silencing of the associated genes but instead disrupts proper expression of those silenced genes under activating conditions. Taken together, these data suggest that Htz1 functions to maintain regulated promoters in a chromatin state that is poised and compatible with transcription.
In contrast to the yeast studies, in vitro and in vivo analyses of H2A.Z function in complex organisms have yielded confounding results. Structural and biophysical analyses have found both stabilizing and destabilizing effects of H2A.Z on the nucleosome structure as well as on the stability of oligonucleosome arrays (1
). In Tetrahymena thermophila
, the H2A.Z equivalent, hv1, is found exclusively in the transcriptionally active macronucleus but not in the transcriptionally silent micronucleus (32
). Drosophila melanogaster
polytene chromosome staining shows a nonrandom distribution of the fly H2A.Z homolog, H2AvD, on both euchromatic and heterochromatic regions (17
). In addition, chromatin immunoprecipitation assays show that H2AvD is localized to transcribing and nontranscribing genes. In trophoblasts and endoderm cells of mouse embryos, H2A.Z is enriched at the pericentric heterochromatin but is depleted on the transcriptionally silenced inactive X chromosome (27
). In vivo cross-linking studies also show that H2A.Z associates with HP1α, a resident protein that marks constitutive heterochromatin in mammalian cells, and in vitro studies using reconstituted nucleosome arrays indicate that H2A.Z and HP1α can synergize to promote chromatin compaction (11
). RNA interference (RNAi) studies have suggested that H2A.Z has a role in chromosome segregation (28
), a function that may be specifically associated with the fraction of H2A.Z detected at centromeres (12
). So far, these studies have focused on the structural functions of mammalian H2A.Z. In this study, we focused on the epigenetic aspects of mammalian H2A.Z and found that it is associated with both euchromatin and facultative heterochromatin. For example, in contrast to mouse embryonic cells, H2A.Z is depleted at pericentric heterochromatin in differentiated mouse and human cells. Consistent with that finding, H2A.Z-containing nucleosomes are enriched for K4-methylated H3 and are reduced for K9-methylated H3 compared to the methylation levels of nucleosomes containing H2A. We also found that a fraction of H2A.Z is monoubiquitylated at the C terminus and that H2A.Z located on the transcriptionally silent inactive X chromosome of female cells is mostly ubiquitylated. These data show a conservation of H2A.Z's function in transcriptional regulation in yeast and mammalian cells and that ubiquitylation of H2A.Z distinguishes the fraction of this variant that is associated with facultative heterochromatin from the euchromatin-associated H2A.Z.