Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity.

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.

N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing.

Post-translational modifications of histones play a key role in DNA-based processes, like transcription, by modulating chromatin structure. N-terminal acetylation is unique among the numerous histone modifications because it is deposited on the N-alpha amino group of the first residue instead of the side-chain of amino acids. The function of this modification and its interplay with other internal histone marks has not been previously addressed. Here, we identified N-terminal acetylation of H4 (N-acH4) as a novel regulator of arginine methylation and chromatin silencing in Saccharomyces cerevisiae. Lack of the H4 N-alpha acetyltransferase (Nat4) activity results specifically in increased deposition of asymmetric dimethylation of histone H4 arginine 3 (H4R3me2a) and in enhanced ribosomal-DNA silencing. Consistent with this, H4 N-terminal acetylation impairs the activity of the Hmt1 methyltransferase towards H4R3 in vitro. Furthermore, combinatorial loss of N-acH4 with internal histone acetylation at lysines 5, 8 and 12 has a synergistic induction of H4R3me2a deposition and rDNA silencing that leads to a severe growth defect. This defect is completely rescued by mutating arginine 3 to lysine (H4R3K), suggesting that abnormal deposition of a single histone modification, H4R3me2a, can impact on cell growth. Notably, the cross-talk between N-acH4 and H4R3me2a, which regulates rDNA silencing, is induced under calorie restriction conditions. Collectively, these findings unveil a molecular and biological function for H4 N-terminal acetylation, identify its interplay with internal histone modifications, and provide general mechanistic implications for N-alpha-terminal acetylation, one of the most common protein modifications in eukaryotes.

Involvement of the chloroplastic isoform of tRNA ligase in the replication of viroids belonging to the family Avsunviroidae.

Avocado sunblotch viroid, peach latent mosaic viroid, chrysanthemum chlorotic mottle viroid, and eggplant latent viroid (ELVd), the four recognized members of the family Avsunviroidae, replicate through the symmetric pathway of an RNA-to-RNA rolling-circle mechanism in chloroplasts of infected cells. Viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length RNAs with 5'-hydroxyl and 2',3'-phosphodiester termini that are subsequently circularized. We report that a recombinant version of the chloroplastic isoform of the tRNA ligase from eggplant (Solanum melongena L.) efficiently catalyzes in vitro circularization of the plus [(+)] and minus [(-)] monomeric linear replication intermediates from the four Avsunviroidae. We also show that while this RNA ligase specifically recognizes the genuine monomeric linear (+) ELVd replication intermediate, it does not do so with five other monomeric linear (+) ELVd RNAs with their ends mapping at different sites along the molecule, despite containing the same 5'-hydroxyl and 2',3'-phosphodiester terminal groups. Moreover, experiments involving transient expression of a dimeric (+) ELVd transcript in Nicotiana benthamiana Domin plants preinoculated with a tobacco rattle virus-derived vector to induce silencing of the plant endogenous tRNA ligase show a significant reduction of ELVd circularization. In contrast, circularization of a viroid replicating in the nucleus occurring through a different pathway is unaffected. Together, these results support the conclusion that the chloroplastic isoform of the plant tRNA ligase is the host enzyme mediating circularization of both (+) and (-) monomeric linear intermediates during replication of the viroids belonging to the family Avsunviroidae.

Cross-talk among epigenetic modifications: lessons from histone arginine methylation.

Epigenetic modifications, including those occurring on DNA and on histone proteins, control gene expression by establishing and maintaining different chromatin states. In recent years, it has become apparent that epigenetic modifications do not function alone, but work together in various combinations, and cross-regulate each other in a manner that diversifies their functional states. Arginine methylation is one of the numerous PTMs (post-translational modifications) occurring on histones, catalysed by a family of PRMTs (protein arginine methyltransferases). This modification is involved in the regulation of the epigenome largely by controlling the recruitment of effector molecules to chromatin. Histone arginine methylation associates with both active and repressed chromatin states depending on the residue involved and the configuration of the deposited methyl groups. The present review focuses on the increasing number of cross-talks between histone arginine methylation and other epigenetic modifications, and describe how these cross-talks influence factor binding to regulate transcription. Furthermore, we present models of general cross-talk mechanisms that emerge from the examples of histone arginine methylation and allude to various techniques that help decipher the interplay among epigenetic modifications.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

A chloroplastic RNA ligase activity analogous to the bacterial and archaeal 2´-5' RNA ligase.

Bacteria and archaea contain a 2'-5' RNA ligase that seals in vitro 2',3'-cyclic phosphodiester and 5'-hydroxyl RNA termini, generating a 2',5'-phosphodiester bond. In our search for an RNA ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA ligase activity whose properties resemble those of the bacterial and archaeal 2'-5' RNA ligase. The spinach chloroplastic RNA ligase recognizes the 5'-hydroxyl and 2',3'-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2',5'-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear initial substrate. Even if its involvement in viroid replication seems unlikely, the identification of a 2'-5' RNA ligase activity in higher plant chloroplasts, with properties very similar to an analogous enzyme widely distributed in bacterial and archaeal proteomes, is intriguing and suggests an important biological role so far unknown.

Monomeric linear RNA of citrus exocortis viroid resulting from processing in vivo has 5'-phosphomonoester and 3'-hydroxyl termini: implications for the RNase and RNA ligase involved in replication.

Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(+)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5' and 3' cDNA ends and in vitro ligation assays, that ml CEVd (+) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5'-phosphomonoester and 3'-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.

Histone Modifications as an Intersection Between Diet and Longevity.

Histone modifications are key epigenetic regulators that control chromatin structure and gene transcription, thereby impacting on various important cellular phenotypes. Over the past decade, a growing number of studies have indicated that changes in various histone modifications have a significant influence on the aging process. Furthermore, it has been revealed that the abundance and localization of histone modifications are responsive to various environmental stimuli, such as diet, which can also affect gene expression and lifespan. This supports the notion that histone modifications can serve as a main cellular platform for signal integration. Hence, in this review we focus on the role of histone modifications during aging, report the data indicating that diet affects histone modification levels and explore the idea that histone modifications may function as an intersection through which diet regulates lifespan. A greater understanding of the epigenetic mechanisms that link environmental signals to longevity may provide new strategies for therapeutic intervention in age-related diseases and for promoting healthy aging.

Beyond the histone tail: acetylation at the nucleosome dyad commands transcription.

Post-translational modifications (PTMs) of histones have been implicated in cellular processes such as transcription, replication and DNA repair. These processes normally involve dynamic changes in chromatin structure and DNA accessibility. Most of the PTMs reported so far map on the histone tails and essentially affect chromatin structure indirectly by recruiting effector proteins. A recent study by Schneider and colleagues published in Cell (1) has uncovered the function of H3K122 acetylation found within the histone globular domain and specifically positioned on the DNA-bound surface of the nucleosome. Their findings demonstrate a direct effect of histone PTMs on chromatin dynamics, and propose that modifications located in different parts of the nucleosome employ distinct regulatory mechanisms.

Viroid replication: rolling-circles, enzymes and ribozymes.

Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5' and 3' termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.

Processing of RNAs of the family Avsunviroidae in Chlamydomonas reinhardtii chloroplasts.

The family Avsunviroidae comprises four viroid species with the ability to form hammerhead ribozymes that mediate self-cleavage of the multimeric plus and minus strands resulting from replication in the chloroplast through a symmetric rolling-circle mechanism. Research on these RNAs is restricted by their host range, which is limited to the plants wherein they were initially identified and some closely related species. Here we report cleavage and ligation in transplastomic Chlamydomonas reinhardtii expressing plus- and minus-strand dimeric transcripts of representative members of the family Avsunviroidae. Despite the absence of viroid RNA-RNA transcription, the C. reinhardtii-based system can be used to address intriguing questions about viroid RNA processing and, in particular, about the cellular factors involved in cleavage and ligation.