Region-specific H3K9me3 gain in aged somatic tissues in Caenorhabditis elegans.

Epigenetic alterations occur as organisms age, and lead to chromatin deterioration, loss of transcriptional silencing and genomic instability. Dysregulation of the epigenome has been associated with increased susceptibility to age-related disorders. In this study, we aimed to characterize the age-dependent changes of the epigenome and, in turn, to understand epigenetic processes that drive aging phenotypes. We focused on the aging-associated changes in the repressive histone marks H3K9me3 and H3K27me3 in C. elegans. We observed region-specific gain and loss of both histone marks, but the changes are more evident for H3K9me3. We further found alteration of heterochromatic boundaries in aged somatic tissues. Interestingly, we discovered that the most statistically significant changes reflected H3K9me3-marked regions that are formed during aging, and are absent in developing worms, which we termed "aging-specific repressive regions" (ASRRs). These ASRRs preferentially occur in genic regions that are marked by high levels of H3K9me2 and H3K36me2 in larval stages. Maintenance of high H3K9me2 levels in these regions have been shown to correlate with a longer lifespan. Next, we examined whether the changes in repressive histone marks lead to de-silencing of repetitive DNA elements, as reported for several other organisms. We observed increased expression of active repetitive DNA elements but not global re-activation of silent repeats in old worms, likely due to the distributed nature of repetitive elements in the C. elegans genome. Intriguingly, CELE45, a putative short interspersed nuclear element (SINE), was greatly overexpressed at old age and upon heat stress. SINEs have been suggested to regulate transcription in response to various cellular stresses in mammals. It is likely that CELE45 RNAs also play roles in stress response and aging in C. elegans. Taken together, our study revealed significant and specific age-dependent changes in repressive histone modifications and repetitive elements, providing important insights into aging biology.

Chromatin Immunoprecipitation from Caenorhabditis elegans Somatic Cells.

Chromatin Immunoprecipitation is a regularly used method to detect DNA-protein interaction in diverse biological samples. Here we describe the application of ChIP for histone modifications in adult-stage Caenorhabditis elegans somatic cells.

Unique patterns of trimethylation of histone H3 lysine 4 are prone to changes during aging in Caenorhabditis elegans somatic cells.

Tri-methylation on histone H3 lysine 4 (H3K4me3) is associated with active gene expression but its regulatory role in transcriptional activation is unclear. Here we used Caenorhabditis elegans to investigate the connection between H3K4me3 and gene expression regulation during aging. We uncovered around 30% of H3K4me3 enriched regions to show significant and reproducible changes with age. We further showed that these age-dynamic H3K4me3 regions largely mark gene-bodies and are acquired during adult stages. We found that these adult-specific age-dynamic H3K4me3 regions are correlated with gene expression changes with age. In contrast, H3K4me3 marking established during developmental stages remained largely stable with age, even when the H3K4me3 associated genes exhibited RNA expression changes during aging. Importantly, the genes associated with changes in H3K4me3 and RNA levels with age are enriched for functional groups commonly implicated in aging biology. Therefore, our findings suggested divergent roles of H3K4me3 in gene expression regulation during aging, with important implications on aging-dependent pathophysiologies.

Trimethylation of Lys36 on H3 restricts gene expression change during aging and impacts life span.

Functional data indicate that specific histone modification enzymes can be key to longevity in Caenorhabditis elegans, but the molecular basis of how chromatin structure modulates longevity is not well understood. In this study, we profiled the genome-wide pattern of trimethylation of Lys36 on histone 3 (H3K36me3) in the somatic cells of young and old Caenorhabditis elegans. We revealed a new role of H3K36me3 in maintaining gene expression stability through aging with important consequences on longevity. We found that genes with dramatic expression change during aging are marked with low or even undetectable levels of H3K36me3 in their gene bodies irrespective of their corresponding mRNA abundance. Interestingly, 3' untranslated region (UTR) length strongly correlates with H3K36me3 levels and age-dependent mRNA expression stability. A similar negative correlation between H3K36me3 marking and mRNA expression change during aging was also observed in Drosophila melanogaster, suggesting a conserved mechanism for H3K36me3 in suppressing age-dependent mRNA expression change. Importantly, inactivation of the methyltransferase met-1 resulted in a decrease in global H3K36me3 marks, an increase in mRNA expression change with age, and a shortened life span, suggesting a causative role of the H3K36me3 marking in modulating age-dependent gene expression stability and longevity.

Phosphorylation of H4 Ser 47 promotes HIRA-mediated nucleosome assembly.

Histone H3 variant H3.3, while differing from canonical H3 (H3.1) by only five amino acids, is assembled into nucleosomes, along with histone H4, at genic regions by the histone chaperone HIRA, whereas H3.1 is assembled into nucleosomes in a CAF-1-dependent reaction. Here, we show that phosphorylation of histone H4 Ser 47 (H4S47ph), catalyzed by the PAK2 kinase, promotes nucleosome assembly of H3.3-H4 and inhibits nucleosome assembly of H3.1-H4 by increasing the binding affinity of HIRA to H3.3-H4 and reducing association of CAF-1 with H3.1-H4. These results reveal a mechanism whereby H4S47ph distinctly regulates nucleosome assembly of H3.1 and H3.3.

Histone H3-K56 acetylation is important for genomic stability in mammals.

Histone H3 lysine 56 acetylation (H3K56Ac) has recently been identified and shown to be important for genomic stability in yeast. However, whether or not H3K56 acetylation occurs in mammals is not clear. Here, we report that H3K56Ac exists in mammals. Mammalian H3K56Ac requires the histone chaperone Asf1 and occurs mainly at the S phase in unstressed cells. Moreover, SIRT1, which is a mammalian member of sirtuin family of NAD(+)-dependent deacetylases, regulates the deacetylation of H3K56. We further showed that proper H3K56 acetylation is critical for genomic stability and DNA damage response. These results establish the existence and functional significance of H3K56Ac in mammals and identify two regulators of this modification.

Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog.

DNA methylation plays an important role in gene silencing in mammals. Two de novo methyltransferases, Dnmt3a and Dnmt3b, are required for the establishment of genomic methylation patterns in development. However, little is known about their coordinate function in the silencing of genes critical for embryonic development and how their activity is regulated. Here we show that Dnmt3a and Dnmt3b are the major components of a native complex purified from embryonic stem cells. The two enzymes directly interact and mutually stimulate each other both in vitro and in vivo. The stimulatory effect is independent of the catalytic activity of the enzyme. In differentiating embryonic carcinoma or embryonic stem cells and mouse postimplantation embryos, they function synergistically to methylate the promoters of the Oct4 and Nanog genes. Inadequate methylation caused by ablating Dnmt3a and Dnmt3b is associated with dysregulated expression of Oct4 and Nanog during the differentiation of pluripotent cells and mouse embryonic development. These results suggest that Dnmt3a and Dnmt3b form a complex through direct contact in living cells and cooperate in the methylation of the promoters of Oct4 and Nanog during cell differentiation. The physical and functional interaction between Dnmt3a and Dnmt3b represents a novel regulatory mechanism to ensure the proper establishment of genomic methylation patterns for gene silencing in development.

Chromatin targeting of de novo DNA methyltransferases by the PWWP domain.

DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both enzymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible for the catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWP-mediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation in satellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.