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SummaryTranscriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in budding yeast to measure how transcriptional bursting changes upon single and double perturbations of chromatin remodeling factors, the transcription factor Gal4 and preinitiation complex (PIC) components. Using dynamic epistasis analysis, we reveal how remodeling of different nucleosomes regulates individual transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC acts synergistically with Gal4 binding to facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. In the absence of remodelers, nucleosomes at canonical TATA boxes are displaced by TBP binding to allow for transcription activation. Overall, our results reveal how promoter nucleosome remodeling, together with transcription factor and PIC binding regulates the kinetics of transcriptional bursting.

Transcription of tRNA genes by RNA Polymerase III (RNAPIII) is tightly regulated by signaling cascades in response to nutrient availability. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulatory factors have not been described. For that reason, we decoded the proteome of a single native tRNA gene locus in yeast. We observed dynamic reprogramming of the core RNAPIII transcription machinery upon nutrient perturbation. In addition, we identified Fpt1, a protein of unknown function. Fpt1 uniquely occupied tRNA genes but its occupancy varied and correlated with the efficiency of RNAPIII eviction upon nutrient perturbation. Decoding the proteome of a tRNA gene in the absence of Fpt1 revealed that Fpt1 promotes eviction of RNAPIII. Cells without Fpt1 also showed impaired shutdown of ribosome biogenesis genes upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and for tuning an integrated physiological response to changing metabolic demands.Abstract Figure

The genome in a eukaryotic cell is packaged into chromatin and regulated by chromatin-binding and chromatin-modifying factors. Many of these factors and their complexes have been identified before, but how each genomic locus interacts with its surrounding proteins in the nucleus over time and in changing conditions remains poorly described. Measuring protein–DNA interactions at a specific locus in the genome is challenging and current techniques such as capture of a locus followed by mass spectrometry require high levels of enrichment. Epi-Decoder, a method developed in budding yeast, enables systematic decoding of the proteome of a single genomic locus of interest without the need for locus enrichment. Instead, Epi-Decoder uses massive parallel chromatin immunoprecipitation of tagged proteins combined with barcoding a genomic locus and counting of coimmunoprecipitated barcodes by DNA sequencing (TAG-ChIP-Barcode-Seq). In this scenario, DNA barcode counts serve as a quantitative readout for protein binding of each tagged protein to the barcoded locus. Epi-Decoder can be applied to determine the protein–DNA interactions at a wide range of genomic loci, such as coding genes, noncoding genes, and intergenic regions. Furthermore, Epi-Decoder provides the option to study protein–DNA interactions upon changing cellular and/or genetic conditions. In this protocol, we describe in detail how to construct Epi-Decoder libraries and how to perform an Epi-Decoder analysis.

Macrophages are critical immune cells in inflammatory diseases, and their differentiation and function are tightly regulated by histone modifications. H3K79 methylation is a histone modification associated with active gene expression, and DOT1L is the only histone methyltransferase for H3K79. Here we determine the role of DOT1L in macrophages by applying a selective DOT1L inhibitor in mouse and human macrophages and using myeloid-specific Dot1l-deficient mice. We found that DOT1L directly regulates macrophage function by controlling lipid biosynthesis gene programs including central lipid regulators like sterol regulatory element-binding proteins SREBP1 and SREBP2. DOT1L inhibition also leads to macrophage hyperactivation, which is associated with disrupted SREBP pathways. In vivo, myeloid Dot1l deficiency reduces atherosclerotic plaque stability and increases the activation of inflammatory plaque macrophages. Our data show that DOT1L is a crucial regulator of macrophage inflammatory responses and lipid regulatory pathways and suggest a high relevance of H3K79 methylation in inflammatory disease.

Spermatozoa have a unique genome organization: their chromatin is almost completely devoid of histones and is formed instead of protamines which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodelling and subsequent reorganization and compaction of spermatozoon genome. Using a mouse model in whichDot1lis knocked-out (KO) in postnatal male germ cells, we found thatDot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomics and transcriptomics analyses performed on spermatids reveal thatDot1l-KO modifies the chromatin prior to histone removal, and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects,Dot1l-KO spermatozoa have less compact heads and are less motile which results in impaired fertility.

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.

The genome in a eukaryotic cell is packaged into chromatin and regulated by chromatin-binding and chromatin-modifying factors. Many of these factors and their complexes have been identified before, but how each genomic locus interacts with its surrounding proteins in the nucleus over time and in changing conditions remains poorly described. Measuring protein-DNA interactions at a specific locus in the genome is challenging and current techniques such as capture of a locus followed by mass spectrometry require high levels of enrichment. Epi-Decoder, a method developed in budding yeast, enables systematic decoding of the proteome of a single genomic locus of interest without the need for locus enrichment. Instead, Epi-Decoder uses massive parallel chromatin immunoprecipitation of tagged proteins combined with barcoding a genomic locus and counting of coimmunoprecipitated barcodes by DNA sequencing (TAG-ChIP-Barcode-Seq). In this scenario, DNA barcode counts serve as a quantitative readout for protein binding of each tagged protein to the barcoded locus. Epi-Decoder can be applied to determine the protein-DNA interactions at a wide range of genomic loci, such as coding genes, noncoding genes, and intergenic regions. Furthermore, Epi-Decoder provides the option to study protein-DNA interactions upon changing cellular and/or genetic conditions. In this protocol, we describe in detail how to construct Epi-Decoder libraries and how to perform an Epi-Decoder analysis.

Cell size varies between cell types but is tightly regulated by cell-intrinsic and extrinsic mechanisms. Cell-size control is important for cell function and changes in cell size are frequently observed in cancer cells. Here we uncover a non-canonical role of SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription regulation, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Although most of SETD2’s cellular functions have been linked to this activity, several non-histone substrates of SETD2 have recently been identified – some of which have been linked to novel functions of SETD2 beyond chromatin regulation. Using multiple, independent perturbation strategies we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that this function is dependent on the catalytic activity of SETD2 but independent of H3K36 methylation. Paradoxically, ectopic overexpression of a decoy SRI domain also increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

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.

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.

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.

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.

The assembly and regulation of chromatin requires coordinated activity of multiple mechanisms. Many factors feed into signaling networks that control the epigenome of a cell. It is this complexity that makes understanding the layers of epigenetic regulation a challenge. Genetic screens have been indispensable for studying chromatin processes. However, they can be laborious and the readout for chromatin changes is often indirect. Epi-ID is a screening strategy in yeast that enables the direct assessment of chromatin status in thousands of gene mutants in parallel. Epi-ID takes advantage of DNA sequences called DNA barcodes that are introduced into a library of yeast knockout mutants at a common chromosomal location in the genome. Chromatin immunoprecipitation on pools of barcoded mutant strains followed by barcode counting by high throughput sequencing will report on the abundance of the chromatin mark of interest in each mutant strain. Epi-ID is applicable to a wide range of chromatin proteins and modifications that are present and can be immunoprecipitated at or around the barcoded region.

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 the genetic interactions of 17 cohesin genes centered on quantitative growth measurements of >52,000 gene pairs in the budding yeast Saccharomyces cerevisiae. 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 help in identifying cohesin interactions relevant in disease etiology., Highlighted Article: The characterization of auxiliary factors in sister chromatid cohesion as identified by genetic interaction mapping in yeast, reveals novel roles for the prefoldin complex and the microtubule-associated protein Irc15.

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 assembly and regulation of chromatin requires coordinated activity of multiple mechanisms. Many factors feed into signaling networks that control the epigenome of a cell. It is this complexity that makes understanding the layers of epigenetic regulation a challenge. Genetic screens have been indispensable for studying chromatin processes. However, they can be laborious and the readout for chromatin changes is often indirect. Epi-ID is a screening strategy in yeast that enables the direct assessment of chromatin status in thousands of gene mutants in parallel. Epi-ID takes advantage of DNA sequences called DNA barcodes that are introduced into a library of yeast knockout mutants at a common chromosomal location in the genome. Chromatin immunoprecipitation on pools of barcoded mutant strains followed by barcode counting by high throughput sequencing will report on the abundance of the chromatin mark of interest in each mutant strain. Epi-ID is applicable to a wide range of chromatin proteins and modifications that are present and can be immunoprecipitated at or around the barcoded region.

Histone modifications regulate key processes of eukaryotic genomes. Misregulation of the enzymes that place these modifications can lead to disease. An example of this is DOT1L, the enzyme that can mono-, di-, and trimethylate the nucleosome core on lysine 79 of histone H3 (H3K79). DOT1L plays a role in development and its misregulation has been implicated in several cancers, most notably leukemias caused by a rearrangement of the MLL gene. A DOT1L inhibitor is in clinical trials for these leukemias and shows promising results, yet we are only beginning to understand DOT1L's function and regulation in the cell. Here, we review what happens upstream and downstream of H3K79 methylation. H3K79 methylation levels are highest in transcribed genes, where H2B ubiquitination can promote DOT1L activity. In addition, DOT1L can be targeted to transcribed regions of the genome by several of its interaction partners. Although methylation levels strongly correlate with transcription, the mechanistic link between the two is unclear and probably context-dependent. Methylation of H3K79 may act through recruiting or repelling effector proteins, but we do not yet know which effectors mediate DOT1L's functions. Understanding DOT1L biology better will help us to understand the effects of DOT1L inhibitors and may allow the development of alternative strategies to target the DOT1L pathway.

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.

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-ChIP-Barcode-Seq technology in budding yeast to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry 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 proteins. Moreover, replication stress induced changes in local chromatin-proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Epi-Decoder will enable the delineation of complex and dynamic protein-DNA interactions across many regions of the genome.

Post-translational protein modification by addition or removal of the small polypeptide ubiquitin is involved in a range of critical cellular processes, like proteasomal protein degradation, DNA repair, gene expression, internalization of membrane proteins, and drug sensitivity. We recently identified genes important for acetaminophen (APAP) toxicity in a comprehensive screen and our findings suggested that a small set of yeast strains carrying deletions of ubiquitin-related genes can be informative for drug toxicity profiling. In yeast, approximately 20 different deubiquitinating enzymes (DUBs) have been identified, of which only one is essential for viability. We investigated whether the toxicity profile of DUB deletion yeast strains would be informative about the toxicological mode of action of APAP. A set of DUB deletion strains was tested for sensitivity and resistance to a diverse series of compounds, including APAP, quinine, ibuprofen, rapamycin, cycloheximide, cadmium, peroxide and amino acids and a cluster analysis was performed. Most DUB deletion strains showed an altered growth pattern when exposed to these compounds by being either more sensitive or more resistant than WT. Toxicity profiling of the DUB strains revealed a remarkable overlap between the amino acid tyrosine and acetaminophen (APAP), but not its stereoisomer AMAP. Furthermore, co-exposure of cells to both APAP and tyrosine showed an enhancement of the cellular growth inhibition, suggesting that APAP and tyrosine have a similar mode of action.

Methylation of histone proteins plays a crucial role controlling genome activity. To understand how the responsible histone methyltransferases are regulated it is important to know their fundamental biochemical properties within the cell and relate these to cellular methylation dynamics. Repeated experiment-modeling cycles have led to insights into the in vivo dynamics of methylation of lysine 79 on histone H3 (H3K79) by the methyltransferase Disruptor of Telomeric Silencing 1 (Dot1). Genetic perturbation in yeast, quantitative measurements, and computational modeling were combined to show that Dot1 employs an uncommon, distributive methylation mechanism. A steady-state in vivo methylation model using this information has provided validated explanations for methylation defects in mutants. Subsequent single-cell models have provided insights into the dynamics of H3K79 methylation throughout the cell cycle and uncovered a role for histone protein aging. These integrated modeling approaches will aid in understanding how regulatory mechanisms influence Dot1’s role in gene expression, cell cycle progression, and cancer and can be applied to other methylation systems.

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.

DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase α at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8.

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.

Post-translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone-deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes-4, whereas trimethylation accumulates toward the 3' end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di- and trimethylation decreases lysine 16 acetylation. Thus di- and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation.