RESUMO
Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.
Assuntos
Processamento Alternativo , Splicing de RNA , Éxons/genética , Íntrons , RNARESUMO
Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.
Assuntos
Poli A , Poliadenilação , Regiões 3' não Traduzidas , Humanos , Poli A/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Zinco/metabolismoRESUMO
The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.
Assuntos
Núcleo Celular , Proteínas de Ligação a RNA , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Splicing de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.
Assuntos
Neurogênese , Splicing de RNA , Processamento Alternativo , Animais , Éxons/genética , Mamíferos , Camundongos , Neurogênese/genética , Neurônios , Proteínas de Ligação a RNA/genéticaRESUMO
Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport, and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive crosstalk between gene regulatory layers takes advantage of dynamic spatial, physical, and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control.
Assuntos
Redes Reguladoras de Genes , Splicing de RNA , Processamento Alternativo , Animais , Núcleo Celular/genética , Cromatina/metabolismo , Humanos , Sequências Reguladoras de Ácido Ribonucleico , Transcrição GênicaRESUMO
Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.
Assuntos
Transtorno Autístico/fisiopatologia , Disfunção Cognitiva/patologia , Fator de Iniciação Eucariótico 4G/fisiologia , Éxons/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neuroblastoma/patologia , Neurônios/patologia , Animais , Comportamento Animal , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neurogênese , Neurônios/metabolismo , Biossíntese de Proteínas , Splicing de RNA , Células Tumorais CultivadasRESUMO
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
Assuntos
Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Antivirais , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Citoesqueleto , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Fosfoproteínas/genética , Transporte Proteico , Proteoma/genética , SARS-CoV-2/genética , Transdução de Sinais , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.
Assuntos
Processamento Alternativo/fisiologia , Engenharia Genética/métodos , Sítios de Splice de RNA/fisiologia , Animais , Transtorno Autístico/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Éxons/fisiologia , Humanos , Camundongos , Proteínas do Tecido Nervoso , Neurogênese , Neurônios , Precursores de RNA/fisiologia , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA , Ribonucleoproteínas , Fatores de Processamento de Serina-Arginina , SpliceossomosRESUMO
Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, we show that the genome is segmented into five principal chromatin types that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. We identify a repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, we provide evidence that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell.
Assuntos
Cromatina/classificação , Proteínas de Ligação a DNA/análise , Proteínas de Drosophila/análise , Drosophila melanogaster/genética , Animais , Linhagem Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Análise de Componente PrincipalRESUMO
Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.
Assuntos
Processamento Alternativo , Redes Reguladoras de Genes , Análise de Sequência de RNA/métodos , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Camundongos , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/genéticaRESUMO
Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.
Assuntos
Processamento Alternativo , Linhagem da Célula , Camadas Germinativas , Proteínas de Ligação a RNA/metabolismo , Diferenciação Celular , Endoderma , Coração , Humanos , MesodermaRESUMO
The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.
Assuntos
Processamento Alternativo , Células-Tronco Embrionárias/patologia , Regulação da Expressão Gênica , Células-Tronco Neurais/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células-Tronco Neurais/patologiaRESUMO
The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. Here we show that a CTD arginine residue (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires protein arginine methyltransferase 5 (PRMT5) and recruits the Tudor domain of the survival of motor neuron (SMN, also known as GEMIN1) protein, which is mutated in spinal muscular atrophy. SMN interacts with senataxin, which is sometimes mutated in ataxia oculomotor apraxia type 2 and amyotrophic lateral sclerosis. Because POLR2A R1810me2s and SMN, like senataxin, are required for resolving RNA-DNA hybrids created by RNA polymerase II that form R-loops in transcription termination regions, we propose that R1810me2s, SMN, and senataxin are components of an R-loop resolution pathway. Defects in this pathway can influence transcription termination and may contribute to neurodegenerative disorders.
Assuntos
Arginina/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Terminação da Transcrição Genética , Linhagem Celular , Dano ao DNA , DNA Helicases , Humanos , Metilação , Enzimas Multifuncionais , Doenças Neurodegenerativas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Elongação da Transcrição GenéticaRESUMO
The vertebrate and neural-specific Ser/Arg (SR)-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells while weakening 3' splice site recognition and contributing to exon skipping in nonneural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis.
Assuntos
Processamento Alternativo , Éxons , Modelos Genéticos , Neurogênese/genética , Animais , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Motivos de NucleotídeosRESUMO
Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.
Assuntos
Processamento Alternativo , Bases de Dados de Ácidos Nucleicos , Éxons , Redes Reguladoras de Genes , Isoformas de Proteínas , Animais , Galinhas , Humanos , Camundongos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genéticaRESUMO
Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-ß (Esrrb). Importantly, overexpression of YY2 directs the differentiation of mESCs into cardiovascular lineages. We show that the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) promotes the retention of an intron in the 5'-UTR of Yy2 mRNA that confers sensitivity to 4E-BP-mediated translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and document a multilayer regulatory mechanism that controls its expression.
Assuntos
Processamento Alternativo/fisiologia , Diferenciação Celular , Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Blastocisto/metabolismo , Proteínas de Transporte/metabolismo , Linhagem da Célula , Autorrenovação Celular/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Íntrons , Camundongos , Camundongos Knockout , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfoproteínas , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Fator de Transcrição YY1/metabolismoRESUMO
Alternative splicing (AS) of precursor RNAs is responsible for greatly expanding the regulatory and functional capacity of eukaryotic genomes. Of the different classes of AS, intron retention (IR) is the least well understood. In plants and unicellular eukaryotes, IR is the most common form of AS, whereas in animals, it is thought to represent the least prevalent form. Using high-coverage poly(A)(+) RNA-seq data, we observe that IR is surprisingly frequent in mammals, affecting transcripts from as many as three-quarters of multiexonic genes. A highly correlated set of cis features comprising an "IR code" reliably discriminates retained from constitutively spliced introns. We show that IR acts widely to reduce the levels of transcripts that are less or not required for the physiology of the cell or tissue type in which they are detected. This "transcriptome tuning" function of IR acts through both nonsense-mediated mRNA decay and nuclear sequestration and turnover of IR transcripts. We further show that IR is linked to a cross-talk mechanism involving localized stalling of RNA polymerase II (Pol II) and reduced availability of spliceosomal components. Collectively, the results implicate a global checkpoint-type mechanism whereby reduced recruitment of splicing components coupled to Pol II pausing underlies widespread IR-mediated suppression of inappropriately expressed transcripts.
Assuntos
Processamento Alternativo , Íntrons/genética , Mamíferos/genética , Transcriptoma/genética , Células 3T3 , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Evolução Molecular , Células HeLa , Humanos , Células K562 , Mamíferos/classificação , Camundongos , Modelos Genéticos , Especificidade de Órgãos , Análise de Componente Principal , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Vertebrados/classificação , Vertebrados/genéticaRESUMO
Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.
Assuntos
Processamento Alternativo , Éxons , Genes Reporter , Ensaios de Triagem em Larga Escala , Luciferases de Vaga-Lume , Bibliotecas de Moléculas Pequenas , Animais , Processamento Alternativo/efeitos dos fármacos , Humanos , Ensaios de Triagem em Larga Escala/métodos , Camundongos , Éxons/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Células HEK293 , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacosRESUMO
Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.