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

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.     

A reexamination of spreading of position-effect variegation in the white-roughest region of Drosophila melanogaster

In Drosophila, heterochromatin causes mosaic silencing of euchromatic genes brought next to it by chromosomal rearrangements. Silencing has been observed to "spread": genes closer to the heterochromatic rearrangement breakpoint are silenced more frequently than genes farther away. We have examined silencing of the white and roughest genes in the variegating rearrangements In(1)w(m4), In(1)w(mMc), and In(1)w(m51b). Eleven stocks bearing these chromosomes differ widely in the strength of silencing of white and roughest. Stock-specific differences in the relative frequencies of inactivation of white and roughest were found that map to the white-roughest region or the adjacent heterochromatin. Most stock-specific differences did not correlate with gross differences in the heterochromatic content of the rearranged chromosomes; however, two stocks, In(1)w(m51b) and In(1)w(mMc), were found to have anomalous additional heterochromatin that may act in trans to suppress variegating alleles. In comparing different stocks, the frequency of silencing of the roughest gene, which is more distant from heterochromatin, does not correlate with the frequency of silencing of the more proximal white gene on the same chromosome, in contradiction to the expectation of models of continuous linear propagation of silencing. We frequently observed rough eye tissue that is pigmented, as though an active white gene is skipped.     

Boundaries of transcriptionally silent chromatin in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, heterochromatic gene silencing has been found within HMR and HML silent mating type loci, the telomeres, and the rRNA-encoding DNA. There may be boundary elements that regulate the spread of silencing to protect genes adjacent to silenced domains from this epigenetic repressive effect. Many assays show that specific DNA regulatory elements separate a euchromatic locus from a neighboring heterochromatic domain and thereby function as a boundary. Alternatively, DNA-independent mechanisms such as competition between acetylated and deacetylated histones are also reported to contribute to gene insulation. However, the mechanism by which boundaries are formed is not clear. Here, the characteristics and functions of boundaries at silenced domains in S. cerevisiae are discussed.     

The physiological functions of prion protein

Foreign DNA can be readily integrated into the genomes of mammalian embryonic cells by retroviral infection, DNA microinjection, and transfection protocols. However, the transgenic DNA is frequently not expressed or is expressed at levels far below expectation. In a number of organisms such as yeast, plants, Drosophila, and nematodes, silencing of transfected genes is triggered by the interaction between adjacent or dispersed copies of genes of identical sequence. We set out to determine whether a mechanism similar to repeat-induced gene silencing (RIGS) is responsible for the silencing of transgenes in murine embryonal carcinoma stem cells. We compared the expression of identical reporter gene constructs in cells carrying single or multiple copies and found that the level of expression per integrated copy was more than 10-fold higher in single-copy integrants. In cells carrying tandem copies of the transgene, many copies were methylated and clones frequently failed to express both copies of near-identical integrated alleles. Addition of extra copies of the reporter gene coding sequence reduced the level of expression from the same reporter driven by a eukaryotic promoter. We also found that inhibitors of histone deacetylase such as trichostatin A forestall the silencing of multicopy transgenes, suggesting that chromatin mediates the silencing of transfected genes. This evidence is consistent with the idea that RIGS does occur in mammalian embryonic stem cells although silencing of single-copy transgenes also occurs, suggesting that RIGS is only one of the mechanisms responsible for triggering transgene silencing.     

Molecular biology of nickel carcinogenesis

A review of the molecular mechanisms of nickel carcinogenesis has been compiled. This work is based upon approximately 20 years of research conducted in my laboratory. Molecular mechanisms of nickel carcinogenesis are considered from the pointofview of the uptake of nickel, both soluble and insoluble particles in cells, its dissolution and its effects on heterochromatin. Molecular mechanisms by which nickel induces gene silencing in cells by DNA hypermethylation in mammalian cells and by inhibiting histone acetylation in yeast cells are also discussed.