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Conventional CD8+ memory T cells develop upon stimulation with foreign antigen and provide increased protection upon re-challenge. Over the past two decades, new subsets of CD8+ T cells have been identified that acquire memory features independently of antigen exposure. These antigen-inexperienced memory T cells (TAIM) are described under several names including innate memory, virtual memory, and memory phenotype. TAIM cells exhibit characteristics of conventional or true memory cells, including antigen-specific responses. In addition, they show responsiveness to innate stimuli and have been suggested to provide additional levels of protection toward infections and cancer. Here, we discuss the current understanding of TAIM cells, focusing on extrinsic and intrinsic molecular conditions that favor their development, their molecular definitions and immunological properties, as well as their transcriptional and epigenetic regulation.

Histone modifying enzymes play critical roles in many key cellular processes and are appealing proteins for targeting by small molecules in disease. However, while the functions of histone modifying enzymes are often linked to epigenetic regulation of the genome, an emerging theme is that these enzymes often also act by non-catalytic and/or non-epigenetic mechanisms. SETD2 (Set2 in yeast) is best known for associating with the transcription machinery and methylating histone H3 on lysine 36 (H3K36) during transcription. This well-characterized molecular function of SETD2 plays a role in fine-tuning transcription, maintaining chromatin integrity, and mRNA processing. Here we give an overview of the various molecular functions and mechanisms of regulation of H3K36 methylation by Set2/SETD2. These fundamental insights are important to understand SETD2’s role in disease, most notably in cancer in which SETD2 is frequently inactivated. SETD2 also methylates non-histone substrates such as α-tubulin which may promote genome stability and contribute to the tumor-suppressor function of SETD2. Thus, to understand its role in disease, it is important to understand and dissect the multiple roles of SETD2 within the cell. In this review we discuss how histone methylation by Set2/SETD2 has led the way in connecting histone modifications in active regions of the genome to chromatin functions and how SETD2 is leading the way to showing that we also have to look beyond histones to truly understand the physiological role of an ‘epigenetic’ writer enzyme in normal cells and in disease.

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell–specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein–protein relationships and protein functions at the chromatin template.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B‐cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B‐cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro‐proliferative, pro‐GC program. In addition, DOT1L indirectly supports the repression of an anti‐proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B‐cell naivety and GC B‐cell differentiation., The histone H3K79 methyltransferase DOT1L plays a central role in B cell development and differentiation. DOT1L maintains B cells naivety by orchestrating critical transcriptional and epigenetic regulators.

17 p.-7 fig., The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. Through an unbiased chromatin immunoprecipitation (ChIP)-based approach called Epigenetics-IDentifier (Epi-ID), we aimed to identify regulators of crotonylation, succinylation and butyrylation in thousands of yeast mutants simultaneously. However, highly correlative results led us to further investigate the specificity of the pan-K-acyl antibodies used in our Epi-ID studies. This revealed cross-reactivity and lack of specificity of pan-K-acyl antibodies in various assays. Our findings suggest that the antibodies might recognize histone acetylation in vivo, in addition to histone acylation, due to the vast overabundance of acetylation compared to other acylation modifications in cells. Consequently, our Epi-ID screen mostly identified factors affecting histone acetylation, including known (e.g. GCN5, HDA1, and HDA2) and unanticipated (MET7, MTF1, CLB3, and RAD26) factors, expanding the repertoire of acetylation regulators. Antibody-independent follow-up experiments on the Gcn5-Ada2-Ada3 (ADA) complex revealed that, in addition to acetylation and crotonylation, ADA has the ability to butyrylate histones. Thus, our Epi-ID screens revealed limits of using pan-K-acyl antibodies in epigenetics research, expanded the repertoire of regulators of histone acetylation, and attributed butyrylation activity to the ADA complex., This work was financially supported by an Investment Grant NWO Medium (Grant Number 91116004, partly financed by ZonMw) to P.A.v.V., an NWO VICI grant (Grant Number 016.130.627) to F.v.L., and an ERC Consolidator grant (Grant Number 617485) to H.v.A.

Significance Cytotoxic CD8+ T cells control infectious diseases as well as cancer. Memory T cells are a subset of CD8+ T cells that have previously encountered and rapidly responded to a specific antigen. Here we identified the epigenetic writer DOT1L as a central regulator of T cell physiology. In the absence of DOT1L, which methylates histone H3K79 at active genes, cytotoxic T cells prematurely acquired memory features without having encountered an antigen. However, loss of DOT1L in the T cell lineage led to a compromised immune response. Our findings highlight the importance of epigenetic signals in regulating T cell differentiation. Understanding these epigenetic signals is important for developing strategies to modulate T cell immunity., Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

In eukaryotes, nucleosomes form a barrier to DNA templated reactions and must be dynamically disrupted to provide access to the genome. During nucleosome (re)assembly, histones can be replaced by new histones, erasing post-translational modifications. Measuring histone turnover in mammalian cells has mostly relied on inducible overexpression of histones, which may influence and distort natural histone deposition rates. We have previously used recombination-induced tag exchange (RITE) to study histone dynamics in budding yeast. RITE is a method to follow protein turnover by genetic switching of epitope tags using Cre recombinase and does not rely on inducible overexpression. Here, we applied RITE to study the dynamics of the replication-independent histone variant H3.3 in human cells. Epitope tag-switching could be readily detected upon induction of Cre-recombinase, enabling the monitoring old and new H3.3 in the same pool of cells. However, the rate of tag-switching was lower than in yeast cells. Analysis of histone H3.3 incorporation by chromatin immunoprecipitation did not recapitulate previously reported aspects of H3.3 dynamics such as high turnover rates in active promoters and enhancers. We hypothesize that asynchronous Cre-mediated DNA recombination in the cell population leads to a low time resolution of the H3.3-RITE system in human cells. We conclude that RITE enables the detection of old and new proteins in human cells and that the time-scale of tag-switching prevents the capture of high turnover events in a population of cells. Instead, RITE might be more suited for tracking long-lived histone proteins in human cells.

Chromatin, the complex of DNA and protein in cell nuclei, can be modified by ubiquitin molecules. It emerges that this modification occurs in a molecular reaction chamber formed from an enzyme and a scaffold protein. A phase-separated reaction chamber for chromatin ubiquitination.

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.

Eukaryotic chromosomes are replicated in interphase and the two newly duplicated sister chromatids are held together by the cohesin complex and several cohesin auxiliary factors. Sister chromatid cohesion is essential for accurate chromosome segregation during mitosis, yet has also been implicated in other processes, including DNA damage repair, transcription and DNA replication. To assess how cohesin and associated factors functionally interconnect and coordinate with other cellular processes, we systematically mapped genetic interactions of 17 cohesin genes centered on quantitative growth measurements of >52,000 gene pairs in budding yeast. Integration of synthetic genetic interactions unveiled a cohesin functional map that constitutes 373 genetic interactions, revealing novel functional connections with post-replication repair, microtubule organization and protein folding. Accordingly, we show that the microtubule-associated protein Irc15 and the prefoldin complex members Gim3, Gim4 and Yke2 are new factors involved in sister chromatid cohesion. Our genetic interaction map thus provides a unique resource for further identification and functional interrogation of cohesin proteins. Since mutations in cohesin proteins have been associated with cohesinopathies and cancer, it may also identify cohesin interactions relevant in disease etiology.

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

CD4+ T cell help is required for the generation of CD8+ cytotoxic T lymphocyte (CTL) memory. Here, we use genome-wide analyses to show how CD4+ T cell help delivered during priming promotes memory differentiation of CTLs. Help signals enhance IL-15-dependent maintenance of central memory T (TCM) cells. More importantly, help signals regulate the size and function of the effector memory T (TEM) cell pool. Helped TEM cells produce Granzyme B and IFNγ upon antigen-independent, innate-like recall by IL-12 and IL-18. In addition, helped memory CTLs express the effector program characteristic of helped primary CTLs upon recall with MHC class I-restricted antigens, likely due to epigenetic imprinting and sustained mRNA expression of effector genes. Our data thus indicate that during priming, CD4+ T cell help optimizes CTL memory by creating TEM cells with innate and help-independent antigen-specific recall capacities., Help from CD4+ T cells is important to induce CD8+ T cell memory responses, but mechanistic insights are lacking. Here, the authors show, by transcriptomics and epigenetics, how CD4+ T cells help program memory CD8+ T cells for help-independent recall by antigens, as well as for innate-like recall responses by IL-12/IL-18 and promoting survival by IL-15.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B cell fate remain unclear. Here we identified a central role for the histone H3K79 methyltransferase DOT1L in controlling B cell differentiation. Murine B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells showed aberrant differentiation and prematurely acquired plasma cell features. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L supports the repression of an anti-proliferative, plasma cell differentiation program by maintaining expression of the H3K27 methyltransferase Ezh2, the catalytic component of Polycomb Repressor Complex 2 (PRC2). Our findings show that DOT1L is a central modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B cell naivety and GC B cell differentiation.

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL‐rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1 Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

Transcription, replication, and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein–DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq) technology in budding yeast. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry (MS) but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 interacting proteins. Moreover, replication stress induced changes in local chromatin proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Finally, we show that native genomic loci can be decoded by efficient construction of barcode libraries assisted by clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Thus, Epi-Decoder is an effective strategy to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus by DNA sequencing., Author summary DNA is packaged by proteins into a structure called chromatin. This packaging plays an important role in gene transcription, DNA replication, and DNA repair. The regulation of these processes is determined by the interplay of proteins that physically interact with specific loci. Although many chromatin proteins have been identified, it remains a challenge to comprehensively identify all the chromatin interactions at any specific locus by protein-based methods such as capture mass spectrometry (MS). Here, we take advantage of DNA barcoding technologies and high-throughput sequencing to tackle this problem. We developed Epi-Decoder, a method that identifies chromatin–protein interaction at a single-copy locus in budding yeast. Epi-Decoder was successfully applied at 3 different genomic loci, resulting in comprehensive overviews of local chromatin interactomes. Most factors identified are known to be involved in chromatin-associated processes such as transcription and DNA replication. We also identified unexpected chromatin–protein interactions. Furthermore, capturing the dynamic chromatin interactome by changing physiological conditions provided insight into the local response to stress. In conclusion, Epi-Decoder is an effective tool for measuring the local proteome of a specific genomic locus by DNA sequencing.

Chromatin not only serves as a packaging material, but also functions as a platform for integrating signals that act upon the genome. Indeed, chromatin is a dynamic macromolecular structure that can be dramatically altered in many ways to facilitate the different transactions at the genome. Examples of such alterations are relocalization of genomic loci within the nucleus upon transcriptional activation or induction of DNA damage, adding or removing post-translational modifications on histones or other chromatin-binding factors, or altering the basic organization of chromatin by moving or removing nucleosomes, i.e. modifying the occupancy of histone octamers. New insights into the scope and mechanisms of chromatin dynamics have recently been obtained by the development of novel techniques to visualize chromatin protein mobility and stability. Here we discuss the developments in this area, with special emphasis on histone exchange, which we define as the replacement of histone proteins without a prerequisi...

Chromatin governs gene regulation and genome maintenance, yet a substantial fraction of the chromatin proteome is still unexplored. Moreover, a global model of the chromatin protein network is lacking. By screening >100 candidates we identify 42 Drosophila proteins that were not previously associated with chromatin, which all display specific genomic binding patterns. Bayesian network modeling of the binding profiles of these and 70 known chromatin components yields a detailed blueprint of the in vivo chromatin protein network. We demonstrate functional compartmentalization of this network, and predict functions for most of the previously unknown chromatin proteins, including roles in DNA replication and repair, and gene activation and repression.

Following mitosis, daughter cells must inherit a functional set of essential proteins and organelles. We applied a genetic tool to simultaneously monitor the kinetics and distribution of old and new proteins marking all intracellular compartments in budding yeasts. Most organelles followed a general pattern whereby preexisting proteins are symmetrically partitioned followed by template-based incorporation of new proteins. Peroxisomes belong to this group, supporting a model of biogenesis by growth and division from preexisting peroxisomes. We detected two exceptions: the nuclear pore complex (NPC) and the spindle pole body (SPB). Old NPCs are stably inherited during successive generations but remained separated from new NPCs, which are incorporated de novo in mother and daughter cells. Only the SPB displayed asymmetrical distribution, with old components primarily inherited by daughter cells and new proteins equally incorporated in both cells. Our analysis resolves conflicting models (peroxisomes, NPC) and reveals unique patterns (NPC, SPB) of organelle inheritance.

Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response.

Contains fulltext : 152512.pdf (Publisher’s version ) (Open Access) Specific degradation of proteins is essential for virtually all cellular processes and is carried out predominantly by the proteasome. The proteasome is important for clearance of damaged cellular proteins. Damaged proteins accumulate over time and excess damaged proteins can aggregate and induce the death of old cells. In yeast, the localization of the proteasome changes dramatically during aging, possibly in response to altered proteasome activity requirements. We followed two key parameters of this process: the distribution of proteasomes in nuclear and cytosolic compartments, and the formation of cytoplasmic aggregate-like structures called proteasome storage granules (PSGs). Whereas replicative young cells efficiently relocalized proteasomes from the nucleus to the cytoplasm and formed PSGs, replicative old cells were less efficient in relocalizing the proteasome and had less PSGs. By using a microscopy-based genome-wide screen, we identified genetic factors involved in these processes. Both relocalization of the proteasome and PSG formation were affected by two of the three N-acetylation complexes. These N-acetylation complexes also had different effects on the longevity of cells, indicating that each N-acetylation complex has different roles in proteasome location and aging.

implicated several additional histone modifiers in life span determination [6,7]. In addition to the organism’s aging pro� gram, stochastic events can also induce epi� genomic changes over time. For example, human monozygotic twins have very similar DNA methylation patterns at birth, but they also show differences that can increase as they get older [8]. These changes may be attributed to environmental in� uences. Another possi� bility is that unavoidable errors occur during the maintenance of epigenetic patterns due to the disruptive nature of transactions at the genome. Transcription by RNA polymerases has the potential to reshuf�e or even erase epi� genetic signals because it requires the transient eviction and subsequent reassembly of histones in the wake of the polymerase. Indeed, several studies suggest that transcription destabilizes chromatin and leads to the partial eviction of (modified) histones and replacement by newly synthesized (unmodified) ones [9]. It is worth noting however, that the introduction of many of the histone modifications in active regions of the genome is promoted by the process of tran� scription initiation or elongation. Therefore, these marks can be maintained even when the histones carrying the marks are evicted [10]. DNA replication is another potential source of epigenetic rearrangements. Ahead of the replication fork, old modified histones dissoci� ate from the DNA and behind the replication fork, histones reassemble on the two daughter strands. The chromatin gaps on the duplicated DNA are complemented by a set of newly synthesized unmodified histones. In order to maintain an epigenetic identity, the cells need to re�establish the modification pattern of the mother cell by modifying the newly synthe� sized histones in the daughter cells. The speed at which this occurs can substantially differ Epigenetic states help maintain cell identity but they are also dynamic entities that respond to signals. Indeed, cells undergoing developmental changes are characterized by global rearrange� ments of the epigenetic landscape. Recent stud� ies suggest that aging is one such epigenome� shifting developmental event. What is more, epigenetic regulators seem to in�uence the aging process. Aging can occur in different contexts. Here we discuss the emerging evidence that both organismal and cellular aging, as well as histone protein aging, have intimate connections to the epigenome. Striking links between organismal aging and epigenome alterations have recently been identi� fied in humans and metazoan model organisms [1]. For example, tissues and cells of aging organ� isms show increased levels or redistribution of heterochromatic marks such as tri �methylation of lysine 9 on histone H3 (H3K9me3) [2,3]. The cause and biological significance of these changes are still unclear; however, a driver for epigenome changes could be an aging pro� gram that changes the expression of chroma� tin�modifying or �demodifying enzymes. For example, loss of H3K27me3 in (prematurely) aging human brains is accompanied by the increased expression of the H3K27�specific demethylase UTX [4]. Aging somatic cells in Caenorhabditis elegans show a similar decline in H3K27me3 and increase in UTX�1 expression [4,5]. Importantly, genetic inactivation of C. elegans UTX�1 prevents the age�induced loss of H3K27me3 and extends life span of the worm via the insulin�signaling pathway, a major life span regulator [4,5]. These findings suggest that loss of H3K27me3, a repressive mark associated with regions of facultative heterochromatin, is not only associated with aging but may in fact be causally involved in the aging process. Recent genetic studies in �ies and worms have

Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated approach to investigate the dynamics of the conserved methylation of histone H3 Lys 79 (H3K79) by Dot1. Our results show that methylation of H3K79 progressively changes after histone deposition, which is incompatible with a rapid copy mechanism. Instead, methylation accumulates on ageing histones, providing the cell with a timer mechanism to directly couple cell-cycle length to changes in chromatin modification on the nucleosome core. © 2011 European Molecular Biology Organization.

Dot1 is a highly conserved methyltransferase that modifies histone H3 on the nucleosome core surface. In contrast to yeast, flies, and humans where a single Dot1 enzyme is responsible for all methylation of H3 lysine 79 (H3K79), African trypanosomes express two DOT1 proteins that methylate histone H3K76 (corresponding to H3K79 in other organisms) in a cell-cycle-regulated manner. Whereas DOT1A is essential for normal cell cycle progression, DOT1B is involved in differentiation and control of antigenic variation of this protozoan parasite. Analysis of DOT1A and DOT1B in trypanosomes or in vitro, to understand how H3K76 methylation is controlled during the cell cycle, is complicated by the lack of genetic tools and biochemical assays. To eliminate these problems, we developed a heterologous expression system in yeast. Whereas Trypanosoma brucei DOT1A predominantly dimethylated H3K79, DOT1B trimethylated H3K79 even in the absence of dimethylation by DOT1A. Furthermore, DOT1A activity was selectively reduced by eliminating ubiquitylation of H2B. The tail of histone H4 was not required for activity of DOT1A or DOT1B. These findings in yeast provide new insights into possible mechanisms of regulation of H3K76 methylation in Trypanosoma brucei.

Understanding disease-associated cellular defects at a molecular level is critical for the development of pharmacological intervention strategies. Recent breakthroughs in microarray and sequencing technologies have provided powerful tools to rapidly reveal the cellular defects caused by alterations in the genome or transcriptome. However, the picture of how the cellular proteome is affected in a disease state and how changes in DNA and RNA affect protein function is often incomplete. This is perhaps not surprising because the functions of proteins are not just determined by primary sequence and abundance, but are under the control of many regulatory mechanisms. Here, we highlight several recent advances in proteomics technologies that are being developed to generate comprehensive human proteome maps and discuss them in the context of strategies that have been developed in simple model organisms. Chemical biology will play a critical role in drafting a map of the proteome with functional information. Chemical genetic approaches that use high-throughput small molecule screening have resulted in the public availability of small molecule datasets through web interfaces such as PubChem. With such approaches, the opportunities to investigate disease and to explore the proteome with chemistry are rapidly increasing. In addition, new tools are being developed to probe protein function. Here we highlight recent developments in chemical biology and the exciting opportunities that are arising with them.

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.

Mouse splenic dendritic cell (DC) subsets possess distinct antigen-presentation abilities. CD8(+) DC are specialized in cross-presentation of antigens to CD8(+) T cells, whereas CD8(-) DC are more efficient in antigen presentation to CD4(+) T cells. In this study, we examined the capacity of CD8(+) and CD8(-) DC subsets to present fungal antigens in MHC class I and II molecules to CD8(+) and CD4(+) T cells, respectively. We used ovalbumin-expressing Saccharomyces cerevisiae (yeast-OVA) as a fungal model system. Both CD8(+) and CD8(-) DC subsets phagocytosed yeast in equal amounts and uptake was mediated via dectin-1. In addition, both DC subsets induced similar OVA-specific CD4(+) T cell proliferation after incubation with yeast-OVA. However, the induction of OVA-specific CD8(+) T cell activation was largely restricted to the CD8(-) DC subset. Furthermore, only CD8(-) DC produced cytokines such as IL-10 and TNF-alpha and increased IL-23p19 and IL-23p40 mRNA levels in response to yeast. Our results strongly suggest that DC subsets have different functions in the elicitation of adaptive immune responses in vivo.

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein ( APP(+1)) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP(+1) proficiently secreted APP(+1). Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP(+1) is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP(+1) level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) ( p

More than 20 residues within the four core histone proteins of the nucleosome are potential sites of post-translational modifications, such as methylation, acetylation, ubiquitination and phosphorylation. It has been hypothesized that specific patterns of these modifications on the nucleosome facilitate recruitment of non-histone proteins to chromatin. When such modifications are restricted to particular regions of the genome, they seem to play an important role in creating specific chromatin domains. However, more recent results suggest that some histone modifications, particularly those that exist on a genome-wide scale, act to reduce nonspecific binding by chromatin proteins involved in silencing. This decrease of promiscuous binding ensures that the silent chromatin proteins are not titrated away from their normal locations on chromosomes. We suggest that preventing such promiscuous binding of chromatin proteins is an important part of generating specificity to create chromatin domains and overall chromosome organization.

Publisher Summary Silencing in Saccharomyces cerevisiae is a chromatin-mediated repression of genes located within specific chromosomal domains. Each time chromosomal DNA replicates, silent chromatin must be recreated by an orchestrated series of assembly processes and events. Mutations affecting DNA replication, cell cycle progression, DNA repair, chromatin assembly, histone modification, telomere length, protein turnover, or cellular metabolism modulate silencing, demonstrating that many cellular pathways are connected to the process. A loss of silencing, particularly at the silent mating loci, also has its consequences. Absence of silencing in a haploid cell triggers a cascade of events that cause the cell to act as if it were a diploid. As a result of its intimate ties to overall cellular physiology and processes, and the fact that silent chromatin is found in all eukaryotes, silencing has become a well-studied aspect of yeast biology. This chapter focuses on genetic and cellular assays to monitor gene silencing in yeast. In addition, strains and vectors most commonly used in the assays are described with their advantages and limitations. The chapter provides an overview is given of assays that can facilitate identification of new silencing components.

beta-D-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. In Trypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of the T. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

1. VP and OT mediate their wealth of effects via 4 receptor subtypes V1a, V1b, V2, and OT receptors. 2. We here review recent insights in the pharmacological properties, structure activity relationships, species differences in ligand specificity, expression patterns, and signal transduction of VP/OT receptor. 3. Furthermore, the existence of additional VP/OT receptor subtypes is discussed.

Background Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome. Results Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1. Conclusions Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.