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.

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.

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.

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.

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma.

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 Hdac1Δ/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.

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.

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.

Yeast PP4 interacts with ATR homolog Ddc2-Mec1 and regulates checkpoint signaling.

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.

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.

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.

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.

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.

Decoding the chromatin proteome of a single 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-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.

A network model of the molecular organization of chromatin in Drosophila.

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.

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.

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism.

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.

The cerebellum in Alzheimer's disease: evaluating its role in cognitive decline.

The cerebellum has long been regarded as essential only for the coordination of voluntary motor activity and motor learning. Anatomical, clinical and neuroimaging studies have led to a paradigm shift in the understanding of the cerebellar role in nervous system function, demonstrating that the cerebellum appears integral also to the modulation of cognition and emotion. The search to understand the cerebellar contribution to cognitive processing has increased interest in exploring the role of the cerebellum in neurodegenerative and neuropsychiatric disorders. Principal among these is Alzheimer's disease. Here we review an already sizeable existing literature on the neuropathological, structural and functional neuroimaging studies of the cerebellum in Alzheimer's disease. We consider these observations in the light of the cognitive deficits that characterize Alzheimer's disease and in so doing we introduce a new perspective on its pathophysiology and manifestations. We propose an integrative hypothesis that there is a cerebellar contribution to the cognitive and neuropsychiatric deficits in Alzheimer's disease. We draw on the dysmetria of thought theory to suggest that this cerebellar component manifests as deficits in modulation of the neurobehavioural deficits. We provide suggestions for future studies to investigate this hypothesis and, ultimately, to establish a comprehensive, causal clinicopathological disease model.

Somatic mutations in neurons during aging and neurodegeneration.

The nervous system is composed of a large variety of neurons with a diverse array of morphological and functional properties. This heterogeneity is essential for the construction and maintenance of a distinct set of neural networks with unique characteristics. Accumulating evidence now indicates that neurons do not only differ at a functional level, but also at the genomic level. These genomic discrepancies seem to be the result of somatic mutations that emerge in nervous tissue during development and aging. Ultimately, these mutations bring about a genetically heterogeneous population of neurons, a phenomenon that is commonly referred to as "somatic brain mosaicism". Improved understanding of the development and consequences of somatic brain mosaicism is crucial to understand the impact of somatic mutations on neuronal function in human aging and disease. Here, we highlight a number of topics related to somatic brain mosaicism, including some early experimental evidence for somatic mutations in post-mitotic neurons of the hypothalamo-neurohypophyseal system. We propose that age-related somatic mutations are particularly interesting, because aging is a major risk factor for a variety of neuronal diseases, including Alzheimer's disease. We highlight potential links between somatic mutations and the development of these diseases and argue that recent advances in single-cell genomics and in vivo physiology have now finally made it possible to dissect the origins and consequences of neuronal mutations in unprecedented detail.

The upstreams and downstreams of H3K79 methylation by DOT1L.

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.