Weiwei Dang

Histone H4 lysine 16 acetylation regulates cellular lifespan.

Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD+-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.

Protein Acetylation Microarray Reveals that NuA4 Controls Key Metabolic Target Regulating Gluconeogenesis

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys514 was crucial for enzymatic activity and the ability of yeast cells to grow on nonfermentable carbon sources. Furthermore, Sir2p deacetylated Pck1p both in vitro and in vivo. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production were dependent on TIP60, the human homolog of ESA1. Our findings demonstrate a regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.

Topography of the ISW2-nucleosome complex: insights into nucleosome spacing and chromatin remodeling

Linker DNA was found to be critical for the specific docking of ISW2 with nucleosomes as shown by mapping the physical contacts of ISW2 with nucleosomes at base‐pair resolution. Hydroxyl radical footprinting revealed that ISW2 not only extensively interacts with the linker DNA, but also approaches the nucleosome from the side perpendicular to the axis of the DNA superhelix and contacts two disparate sites on the nucleosomal DNA from opposite sides of the superhelix. The topography of the ISW2–nucleosome was further delineated by finding which of the ISW2 subunits are proximal to specific sites within the linker and nucleosomal DNA regions by site‐directed DNA photoaffinity labeling. Although ISW2 was shown to contact ∼63 bp of linker DNA, a minimum of 20 bp of linker DNA was required for stable binding of ISW2 to nucleosomes. The remaining ∼43 bp of flanking linker DNA promoted more efficient binding under competitive binding conditions and was functionally important for enhanced sliding of nucleosomes when ISW2 was significantly limiting.

Domain architecture of the catalytic subunit in the ISW2-nucleosome complex.

ABSTRACT ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes.

MYST protein acetyltransferase activity requires active site lysine autoacetylation

The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell‐cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X‐ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1‐K262 and hMOF‐K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

H3K36 methylation promotes longevity by enhancing transcriptional fidelity

Epigenetic mechanisms, including histone post-translational modifications, control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging phenomenon of shortened life span, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a life span screen in Saccharomyces cerevisiae that is designed to identify amino acid residues of histones that regulate yeast replicative aging. Our results reveal that lack of sustained histone H3K36 methylation is commensurate with increased cryptic transcription in a subset of genes in old cells and with shorter life span. In contrast, deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes, suppresses cryptic transcript initiation, and extends life span. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to loss of transcriptional precision that is detrimental to life span, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity.

Regulation of ISW2 by Concerted Action of Histone H4 Tail and Extranucleosomal DNA

ABSTRACT The stable contact of ISW2 with nucleosomal DNA ∼20 bp from the dyad was shown by DNA footprinting and photoaffinity labeling using recombinant histone octamers to require the histone H4 N-terminal tail. Efficient ISW2 remodeling also required the H4 N-terminal tail, although the lack of the H4 tail can be mostly compensated for by increasing the incubation time or concentration of ISW2. Similarly, the length of extranucleosomal DNA affected the stable contact of ISW2 with this same internal nucleosomal site, with the optimal length being 70 to 85 bp. These data indicate the histone H4 tail, in concert with a favorable length of extranucleosomal DNA, recruits and properly orients ISW2 onto the nucleosome for efficient nucleosome remodeling. One consequence of this property of ISW2 is likely its previously observed nucleosome spacing activity.

Inactivation of yeast Isw2 chromatin remodeling enzyme mimics longevity effect of calorie restriction via induction of genotoxic stress response.

ATP-dependent chromatin remodeling is involved in all DNA transactions and is linked to numerous human diseases. We explored functions of chromatin remodelers during cellular aging. Deletion of ISW2, or mutations inactivating the Isw2 enzyme complex, extends yeast replicative lifespan. This extension by ISW2 deletion is epistatic to the longevity effect of calorie restriction (CR), and this mechanism is distinct from suppression of TOR signaling by CR. Transcriptome analysis indicates that isw2Δ partially mimics an upregulated stress response in CR cells. In particular, isw2Δ cells show an increased response to genotoxic stresses, and the DNA repair enzyme Rad51 is important for isw2Δ-mediated longevity. We show that lifespan is also extended in C. elegans by reducing levels of athp-2, a putative ortholog of Itc1/ACF1, a critical subunit of the enzyme complex. Our findings demonstrate that the ISWI class of ATP-dependent chromatin remodeling complexes plays a conserved role during aging and in CR.

Inactivation of the Sas2 histone acetyltransferase delays senescence driven by telomere dysfunction

Changes in telomere chromatin have been linked to cellular senescence, but the underlying mechanisms and impact on lifespan are unclear. We found that inactivation of the Sas2 histone acetyltransferase delays senescence in Saccharomyces cerevisiae telomerase (tlc1) mutants through a homologous recombination‐dependent mechanism. Sas2 acetylates histone H4 lysine 16 (H4K16), and telomere shortening in tlc1 mutants was accompanied by a selective and Sas2‐dependent increase in subtelomeric H4K16 acetylation. Further, mutation of H4 lysine 16 to arginine, which mimics constitutively deacetylated H4K16, delayed senescence and was epistatic to sas2 deletion, indicating that deacetylated H4K16 mediates the delay caused by sas2 deletion. Sas2 normally prevents the Sir2/3/4 heterochromatin complex from leaving the telomere and spreading to internal euchromatic loci. Senescence was delayed by sir3 deletion, but not sir2 deletion, indicating that senescence delay is mediated by release of Sir3 specifically from the telomere repeats. In contrast, sir4 deletion sped senescence and blocked the delay conferred by sas2 or sir3 deletion. We thus show that manipulation of telomere chromatin modulates senescence caused by telomere shortening.

Histone methylation and aging: Lessons learned from model systems

Aging induces myriad cellular and, ultimately, physiological changes that cause a decline in an organisms functional capabilities. Although the aging process and the pathways that regulate it have been extensively studied, only in the last decade have we begun to appreciate that dynamic histone methylation may contribute to this process. In this review, we discuss recent work implicating histone methylation in aging. Loss of certain histone methyltransferases and demethylases changes lifespan in invertebrates, and alterations in histone methylation in aged organisms regulate lifespan and aging phenotypes, including oxidative stress-induced hormesis in yeast, insulin signaling in Caenorhabiditis elegans and mammals, and the senescence-associated secretory phenotype in mammals. In all cases where histone methylation has been shown to impact aging and aging phenotypes, it does so by regulating transcription, suggesting that this is a major mechanism of its action in this context. Histone methylation additionally regulates or is regulated by other cellular pathways that contribute to or combat aging. Given the numerous processes that regulate aging and histone methylation, and are in turn regulated by them, the role of histone methylation in aging is almost certainly underappreciated.