RESUMO
The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.
Assuntos
Bioquímica/métodos , Técnicas de Química Analítica/métodos , Epigenômica/métodos , Epigenoma , Transferência Ressonante de Energia de Fluorescência , Heterocromatina/genética , Histonas/metabolismo , Técnicas de Sonda Molecular , Biossíntese de Proteínas , Fatores de Transcrição/genética , UbiquitinaçãoRESUMO
DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.
Assuntos
Complexos Multienzimáticos/metabolismo , Nucleossomos/enzimologia , Oxytricha/enzimologia , Proteínas de Protozoários/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Tetrahymena thermophila/enzimologia , Complexos Multienzimáticos/genética , Nucleossomos/genética , Oxytricha/genética , Proteínas de Protozoários/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Tetrahymena thermophila/genéticaRESUMO
Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Proteína SMARCB1/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Elementos Facilitadores Genéticos/genética , Feminino , Genoma Humano , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Proteína SMARCB1/química , Proteína SMARCB1/metabolismoRESUMO
Genetic and genomic techniques have proven incredibly powerful for identifying and studying molecular players implicated in the epigenetic regulation of DNA-templated processes such as transcription. However, achieving a mechanistic understanding of how these molecules interact with chromatin to elicit a functional output is non-trivial, owing to the tremendous complexity of the biochemical networks involved. Advances in protein engineering have enabled the reconstitution of 'designer' chromatin containing customized post-translational modification patterns, which, when used in conjunction with sophisticated biochemical and biophysical methods, allow many mechanistic questions to be addressed. In this Review, we discuss how such tools complement established 'omics' techniques to answer fundamental questions on chromatin regulation, focusing on chromatin mark establishment and protein-chromatin interactions.
Assuntos
Cromatina , Epigênese Genética , Cromatina/genética , Histonas/genética , Processamento de Proteína Pós-Traducional , Replicação do DNARESUMO
Work over the last decade has uncovered a new layer of epigenetic dysregulation. It is now appreciated that somatic missense mutations in histones, the packaging agents of genomic DNA, are often associated with human pathologies, especially cancer. Although some of these "oncohistone" mutations are thought to be key drivers of cancer, the impacts of the majority of them on disease onset and progression remain to be elucidated. Here, we survey this rapidly expanding research field with particular emphasis on how histone mutants, even at low dosage, can corrupt chromatin states. This work is unveiling the remarkable intricacies of epigenetic control mechanisms. Throughout, we highlight how studies of oncohistones have leveraged, and in some cases fueled, the advances in our ability to manipulate and interrogate chromatin at the molecular level.
Assuntos
Epigênese Genética , Neoplasias , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologiaRESUMO
Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.
Assuntos
Núcleo Celular , Cromatina , Reagentes de Ligações Cruzadas , Humanos , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/análise , Reagentes de Ligações Cruzadas/química , Transferência de Energia , Epigenômica , Inteínas , Irídio , Mutação , Neoplasias/genética , Fármacos Fotossensibilizantes , Ligação Proteica , Mapas de Interação de ProteínasRESUMO
The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Células HCT116 , Código das Histonas , Histonas/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Transcrição GênicaRESUMO
The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA polymerase II (Pol II) passage through chromatin, yet it is not clear how they cooperate mechanistically. We used genomics, genetic, biochemical, and microscopic approaches to dissect their interplay in Schizosaccharomyces pombe. We show that FACT and H2Bub globally repress antisense transcripts near the 5' end of genes and inside gene bodies, respectively. The accumulation of these transcripts is accompanied by changes at genic nucleosomes and Pol II redistribution. H2Bub is required for FACT activity in genic regions. In the H2Bub mutant, FACT binding to chromatin is altered and its association with histones is stabilized, which leads to the reduction of genic nucleosomes. Interestingly, FACT depletion globally restores nucleosomes in the H2Bub mutant. Moreover, in the absence of Pob3, the FACT Spt16 subunit controls the 3' end of genes. Furthermore, FACT maintains nucleosomes in subtelomeric regions, which is crucial for their compaction.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , UbiquitinaçãoRESUMO
The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.
RESUMO
Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.
Assuntos
Antígenos Nucleares/metabolismo , Histonas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Código das Histonas , Histonas/química , Humanos , Modelos Moleculares , Nucleossomos/química , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , XenopusRESUMO
Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.
Assuntos
Linhagem da Célula , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Mutação com Ganho de Função , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Camundongos , Néfrons/metabolismo , Néfrons/patologia , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein neighborhoods to be mapped using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to the labeling of nearby proteins with cell-permeable probes. Mechanistic studies reveal different labeling pathways are operational depending on the nature of the exogenous probe that is employed. When combined with quantitative proteomics, this photoproximity labeling system generates "snapshots" of protein territories with high temporal and spatial resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein. The technology should be broadly useful in the biomedical area.
Assuntos
Luz , Proteínas , HumanosRESUMO
Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.
Assuntos
Engenharia de Proteínas/métodos , Processamento de Proteína , Inteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Mutations in epigenetic pathways are common oncogenic drivers. Histones, the fundamental substrates for chromatin-modifying and remodelling enzymes, are mutated in tumours including gliomas, sarcomas, head and neck cancers, and carcinosarcomas. Classical 'oncohistone' mutations occur in the N-terminal tail of histone H3 and affect the function of polycomb repressor complexes 1 and 2 (PRC1 and PRC2). However, the prevalence and function of histone mutations in other tumour contexts is unknown. Here we show that somatic histone mutations occur in approximately 4% (at a conservative estimate) of diverse tumour types and in crucial regions of histone proteins. Mutations occur in all four core histones, in both the N-terminal tails and globular histone fold domains, and at or near residues that contain important post-translational modifications. Many globular domain mutations are homologous to yeast mutants that abrogate the need for SWI/SNF function, occur in the key regulatory 'acidic patch' of histones H2A and H2B, or are predicted to disrupt the H2B-H4 interface. The histone mutation dataset and the hypotheses presented here on the effect of the mutations on important chromatin functions should serve as a resource and starting point for the chromatin and cancer biology fields in exploring an expanding role of histone mutations in cancer.
Assuntos
Transformação Celular Neoplásica/genética , Histonas/genética , Mutação/genética , Neoplasias/genética , Histonas/química , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Neoplasias/patologia , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Domínios Proteicos/genética , Processamento de Proteína Pós-TraducionalRESUMO
Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.
Assuntos
Regulação da Expressão Gênica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Serotonina/metabolismo , Fator de Transcrição TFIID/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Feminino , Proteínas de Ligação ao GTP/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Neurônios Serotoninérgicos/citologia , Transglutaminases/metabolismoRESUMO
In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.
Assuntos
Proteínas de Bactérias , Proteínas de Membrana , Peptídeo Hidrolases , Feromônios , Percepção de Quorum , Staphylococcus aureus , Transativadores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Proteínas de Membrana/fisiologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/fisiologia , Feromônios/biossíntese , Percepção de Quorum/genética , Staphylococcus aureus/patogenicidade , Transativadores/genética , Transativadores/metabolismo , VirulênciaRESUMO
Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells. Biochemical studies reveal that the formation of higher-order chromatin structures associated with heterochromatin impose a steric barrier that is refractory to TGM2-mediated histone monoaminylation. A series of structure-activity relationship studies, including the use of DNA-barcoded nucleosome libraries, shows that steric hindrance also steers TGM2 activity at the nucleosome level, restricting monoaminylation to accessible sites within histone tails. Collectively, our data indicate that the activity of TGM2 on chromatin is dictated by substrate accessibility rather than by primary sequence determinants or by the existence of preexisting PTMs, as is the case for many other histone-modifying enzymes.
Assuntos
Histonas , Nucleossomos , Animais , Histonas/genética , Histonas/química , Glutamina , Heterocromatina , Proteína 2 Glutamina gama-Glutamiltransferase , Cromatina/genética , DNA/química , MamíferosRESUMO
H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.
Assuntos
Histonas/genética , Histonas/metabolismo , Ativação Transcricional , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , DNA Polimerase II/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteínas Nucleares/metabolismo , Fatores de Transcrição , UbiquitinaçãoRESUMO
DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.