10th NTES Conference: Nickel and arsenic compounds alter the epigenome of peripheral blood mononuclear cells

The mechanisms that underlie metal carcinogenesis are the subject of intense investigation; however, data from in vitro and in vivo studies are starting to piece together a story that implicates epigenetics as a key player. Data from our lab has shown that nickel compounds inhibit dioxygenase enzymes by displacing iron in the active site. Arsenic is hypothesized to inhibit these enzymes by diminishing ascorbate levels – an important co-factor for dioxygenases. Inhibition of histone demethylase dioxygenases can increase histone methylation levels, which also may affect gene expression. Recently, our lab conducted a series of investigations in human subjects exposed to high levels of nickel or arsenic compounds. Global levels of histone modifications in peripheral blood mononuclear cells (PBMCs) from exposed subjects were compared to low environmentally exposed controls. Results showed that nickel increased H3K4me3 and decreased H3K9me2 globally. Arsenic increased H3K9me2 and decreased H3K9ac globally. Other histone modifications affected by arsenic were sex-dependent. Nickel affected the expression of 2756 genes in human PBMCs and many of the genes were involved in immune and carcinogenic pathways. This review will describe data from our lab that demonstrates for the first time that nickel and arsenic compounds affect global levels of histone modifications and gene expression in exposed human populations.     

Arsenic alters global histone modifications in lymphocytes in vitro and in vivo

Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 μg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 μM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1—proteins that are part of a gene expression silencing complex.     

Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing

We have previously reported that carcinogenic nickel compounds decreased global histone H4 acetylation and silenced the gpt transgene in G12 Chinese hamster cells. However, the nature of this silencing is still not clear. Here, we report that nickel ion exposure increases global H3K9 mono- and dimethylation, both of which are critical marks for DNA methylation and long-term gene silencing. In contrast to the up-regulation of global H3K9 dimethylation, nickel ions decreased the expression and activity of histone H3K9 specific methyltransferase G9a. Further investigation demonstrated that nickel ions interfered with the removal of histone methylation in vivo and directly decreased the activity of a Fe(II)-2-oxoglutarate-dependent histone H3K9 demethylase in nuclear extract in vitro. These results are the first to show a histone H3K9 demethylase activity dependent on both iron and 2-oxoglutarate. Exposure to nickel ions also increased H3K9 dimethylation at the gpt locus in G12 cells and repressed the expression of the gpt transgene. An extended nickel ion exposure led to increased frequency of the gpt transgene silencing, which was readily reversed by treatment with DNA-demethylating agent 5-aza-2'-deoxycytidine. Collectively, our data strongly indicate that nickel ions induce transgene silencing by increasing histone H3K9 dimethylation, and this effect is mediated by the inhibition of H3K9 demethylation.     

On your histone mark,SET, methylate!

Lysine methylation of histones and non-histone proteins has emerged in recent years as a posttranslational modification with wide-ranging cellular implications beyond epigenetic regulation. The molecular interactions between lysine methyltransferases and their substrates appear to be regulated by posttranslational modifications surrounding the lysine methyl acceptor. Two very interesting examples of this cross-talk between methyl-lysine sites are found in the SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain-containing lysine methyltransferases SET7 and SETDB1, whereby the histone H3 trimethylated on lysine 4 (H3K4^me3) modification prevents methylation by SETDB1 on H3 lysine 9 (H3K9) and the histone H3 trimethylated on lysine 9 (H3K9^me3) modification prevents methylation by SET7 on H3K4. A similar cross-talk between posttranslational modifications regulates the functions of non-histone proteins such as the tumor suppressor p53 and the DNA methyltransferase DNMT1. Herein, in cis effects of acetylation, phosphorylation, as well as arginine and lysine methylation on lysine methylation events will be discussed.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Associations between post translational histone modifications,myelomeningocele risk,environmental arsenic exposure,and folate deficiency among participants in a case control study in Bangladesh

Arsenic exposure may contribute to disease risk in humans through alterations in the epigenome. Previous studies reported that arsenic exposure is associated with changes in plasma histone concentrations. Posttranslational histone modifications have been found to differ between the brain tissue of human embryos with neural tube defects and that of controls. Our objectives were to investigate the relationships between plasma histone 3 levels, history of having an infant with myelomeningocele, biomarkers of arsenic exposure, and maternal folate deficiency. These studies took place in Bangladesh, a country with high environmental arsenic exposure through contaminated drinking water. We performed ELISA assays to investigate plasma concentration of total histone 3 (H3) and the histone modification H3K27me3. The plasma samples were collected from 85 adult women as part of a case-control study of arsenic and myelomeningocele risk in Bangladesh. We found significant associations between plasma %H3K27me3 levels and risk of myelomeningocele (P<0.05). Mothers with higher %H3K27me3 in their plasma had lower risk of having an infant with myelomeningocele (odds ratio: 0.91, 95% confidence interval: 0.84, 0.98). We also found that arsenic exposure, as estimated by arsenic concentration in toenails, was associated with lower total H3 concentrations in plasma, but only among women with folate deficiency (β = ?9.99, standard error = 3.91, P=0.02). Our results suggest that %H3K27me3 in maternal plasma differs between mothers of infants with myelomeningocele and mothers of infants without myelomeningocele, and may be a marker for myelomeningocele risk. Women with folate deficiency may be more susceptible to the epigenetic effects of environmental arsenic exposure.     

Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination.     

PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state

Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.     

Genome-wide investigation of the dynamic changes of epigenome modifications after global DNA methylation editing

Chromatin properties are regulated by complex networks of epigenome modifications. Currently, it is unclear how these modifications interact and if they control downstream effects such as gene expression. We employed promiscuous chromatin binding of a zinc finger fused catalytic domain of DNMT3A to introduce DNA methylation in HEK293 cells at many CpG islands (CGIs) and systematically investigated the dynamics of the introduced DNA methylation and the consequent changes of the epigenome network. We observed efficient methylation at thousands of CGIs, but it was unstable at about 90% of them, highlighting the power of genome-wide molecular processes that protect CGIs against DNA methylation. Partially stable methylation was observed at about 1000 CGIs, which showed enrichment in H3K27me3. Globally, the introduced DNA methylation strongly correlated with a decrease in gene expression indicating a direct effect. Similarly, global but transient reductions in H3K4me3 and H3K27ac were observed after DNA methylation but no changes were found for H3K9me3 and H3K36me3. Our data provide a global and time-resolved view on the network of epigenome modifications, their connections with DNA methylation and the responses triggered by artificial DNA methylation revealing a direct repressive effect of DNA methylation in CGIs on H3K4me3, histone acetylation, and gene expression.     

Origins and formation of histone methylation across the human cell cycle

The connections between various nuclear processes and specific histone posttranslational modifications are dependent to a large extent on the acquisition of those modifications after histone synthesis. The reestablishment of histone posttranslational modifications after S phase is especially critical for H3K9 and H3K27 trimethylation, both of which are linked with epigenetic memory and must be stably transmitted from one cellular generation to the next. This report uses a proteomic strategy to interrogate how and when the cell coordinates the formation of histone posttranslational modifications during division. Paramount among the findings is that H3K9 and H3K27 trimethylation begins during S phase but is completed only during the subsequent G(1) phase via two distinct pathways from the unmodified and preexisting dimethylated states. In short, we have systematically characterized the temporal origins and methylation pathways for histone posttranslational modifications during the cell cycle.     

Effects of chronic exposure to arsenic and estrogen on epigenetic regulatory genes expression and epigenetic code in human prostate epithelial cells

Chronic exposures to arsenic and estrogen are known risk factors for prostate cancer. Though the evidence suggests that exposure to arsenic or estrogens can disrupt normal DNA methylation patterns and histone modifications, the mechanisms by which these chemicals induce epigenetic changes are not fully understood. Moreover, the epigenetic effects of co-exposure to these two chemicals are not known. Therefore, the objective of this study was to evaluate the effects of chronic exposure to arsenic and estrogen, both alone and in combination, on the expression of epigenetic regulatory genes, their consequences on DNA methylation, and histone modifications. Human prostate epithelial cells, RWPE-1, chronically exposed to arsenic and estrogen alone and in combination were used for analysis of epigenetic regulatory genes expression, global DNA methylation changes, and histone modifications at protein level. The result of this study revealed that exposure to arsenic, estrogen, and their combination alters the expression of epigenetic regulatory genes and changes global DNA methylation and histone modification patterns in RWPE-1 cells. These changes were significantly greater in arsenic and estrogen combination treated group than individually treated group. The findings of this study will help explain the epigenetic mechanism of arsenic- and/or estrogen-induced prostate carcinogenesis.