The choreography of chromatin in RNA polymerase III regulation.

Regulation of eukaryotic gene expression involves a dynamic interplay between the core transcriptional machinery, transcription factors, and chromatin organization and modification. While this applies to transcription by all RNA polymerase complexes, RNA polymerase III (RNAPIII) seems to be atypical with respect to its mechanisms of regulation. One distinctive feature of most RNAPIII transcribed genes is that they are devoid of nucleosomes, which relates to the high levels of transcription. Moreover, most of the regulatory sequences are not outside but within the transcribed open chromatin regions. Yet, several lines of evidence suggest that chromatin factors affect RNAPIII dynamics and activity and that gene sequence alone does not explain the observed regulation of RNAPIII. Here we discuss the role of chromatin modification and organization of RNAPIII transcribed genes and how they interact with the core transcriptional RNAPIII machinery and regulatory DNA elements in and around the transcribed genes.

Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.

Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.

Altered ubiquitin signaling induces Alzheimer's disease-like hallmarks in a three-dimensional human neural cell culture model.

Alzheimer's disease (AD) is characterized by toxic protein accumulation in the brain. Ubiquitination is essential for protein clearance in cells, making altered ubiquitin signaling crucial in AD development. A defective variant, ubiquitin B + 1 (UBB+1), created by a non-hereditary RNA frameshift mutation, is found in all AD patient brains post-mortem. We now detect UBB+1 in human brains during early AD stages. Our study employs a 3D neural culture platform derived from human neural progenitors, demonstrating that UBB+1 alone induces extracellular amyloid-ß (Aß) deposits and insoluble hyperphosphorylated tau aggregates. UBB+1 competes with ubiquitin for binding to the deubiquitinating enzyme UCHL1, leading to elevated levels of amyloid precursor protein (APP), secreted Aß peptides, and Aß build-up. Crucially, silencing UBB+1 expression impedes the emergence of AD hallmarks in this model system. Our findings highlight the significance of ubiquitin signalling as a variable contributing to AD pathology and present a nonclinical platform for testing potential therapeutics.

Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting.

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

DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

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 remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-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. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-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.

DOT1L regulates lipid biosynthesis and inflammatory responses in macrophages and promotes atherosclerotic plaque stability.

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.

DOT1L inhibition does not modify the sensitivity of cutaneous T cell lymphoma to pan-HDAC inhibitors in vitro.

Cutaneous T-cell lymphomas (CTCLs) are a subset of T-cell malignancies presenting in the skin. The treatment options for CTCL, in particular in advanced stages, are limited. One of the emerging therapies for CTCL is treatment with histone deacetylase (HDAC) inhibitors. We recently discovered an evolutionarily conserved crosstalk between HDAC1, one of the targets of HDAC inhibitors, and the histone methyltransferase DOT1L. HDAC1 negatively regulates DOT1L activity in yeast, mouse thymocytes, and mouse thymic lymphoma. Here we studied the functional relationship between HDAC inhibitors and DOT1L in two human CTCL cell lines, specifically addressing the question whether the crosstalk between DOT1L and HDAC1 observed in mouse T cells plays a role in the therapeutic effect of clinically relevant broad-acting HDAC inhibitors in the treatment of human CTCL. We confirmed that human CTCL cell lines were sensitive to treatment with pan-HDAC inhibitors. In contrast, the cell lines were not sensitive to DOT1L inhibitors. Combining both types of inhibitors did neither enhance nor suppress the inhibitory effect of HDAC inhibitors on CTCL cells. Thus our in vitro studies suggest that the effect of commonly used pan-HDAC inhibitors in CTCL cells relies on downstream effects other than DOT1L misregulation.

The histone methyltransferase SETD2 negatively regulates cell size.

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. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, 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. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain 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.

SETD2: from chromatin modifier to multipronged regulator of the genome and beyond.

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.

Epi-Decoder: Decoding the Local Proteome of a Genomic Locus by Massive Parallel Chromatin Immunoprecipitation Combined with DNA-Barcode Sequencing.

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.

Signals for antigen-independent differentiation of memory CD8+ T cells.

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.

Epigenetics Identifier screens reveal regulators of chromatin acylation and limited specificity of acylation antibodies.

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.

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation.

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 methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells.

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.

Expression of Mutant Ubiquitin and Proteostasis Impairment in Kii Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex Brains.

Kii amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder that is endemic to the Kii peninsula of Japan. The disorder is clinically characterized by a variable combination of parkinsonism, dementia, and motor neuron symptoms. Despite extensive investigations, the etiology and pathogenesis of ALS/PDC remain unclear. At the neuropathological level, Kii ALS/PDC is characterized by neuronal loss and tau-dominant polyproteinopathy. Here, we report the accumulation of several proteins involved in protein homeostasis pathways, that is, the ubiquitin-proteasome system and the autophagy-lysosome pathway, in postmortem brain tissue from a number of Kii ALS/PDC cases (n = 4). Of particular interest is the presence of a mutant ubiquitin protein (UBB+1), which is indicative of disrupted ubiquitin homeostasis. The findings suggest that abnormal protein aggregation is linked to impaired protein homeostasis pathways in Kii ALS/PDC.

Correction to: Somatic mutations in neurons during aging and neurodegeneration.

In the original article, the panels "Brain organoids" and "Transgenics" were included in Fig. 5 without permission.

A genetic interaction map centered on cohesin reveals auxiliary factors involved in sister chromatid cohesion in S. cerevisiae.

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.

Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells.

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.

Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes.

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.