RESUMEN
Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína/metabolismo , Factores de Transcripción/metabolismo , Terminación de la Transcripción Genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Proteínas de Drosophila/química , Drosophila melanogaster , Regulación de la Expresión Génica , Sitios Genéticos , Humanos , Fosforilación , Regiones Promotoras Genéticas , Subunidades de Proteína/química , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Transducción de Señal , Especificidad por Sustrato , Factores de Transcripción/químicaRESUMEN
The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25- to 60-nt-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, 15%-20% of genes display highly unstable promoter Pol II, suggesting that paused polymerase might dissociate from template DNA at these promoters and release a short, non-productive mRNA. Here, we report that paused Pol II can be actively destabilized by the Integrator complex. Specifically, we present evidence that Integrator utilizes its RNA endonuclease activity to cleave nascent RNA and drive termination of paused Pol II. These findings uncover a previously unappreciated mechanism of metazoan gene repression, akin to bacterial transcription attenuation, wherein promoter-proximal Pol II is prevented from entering productive elongation through factor-regulated termination.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Elongación de la Transcripción Genética , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , ARN Polimerasa II/genética , ARN Mensajero/genéticaRESUMEN
mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Nucleares/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , Transcripción Genética , Adenosina/genética , Regulación de la Expresión Génica/genética , Humanos , Metilación , Metiltransferasas/genética , Fosforilación , Transcriptoma/genéticaRESUMEN
Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Sirtuinas/metabolismo , Elongación de la Transcripción Genética , Acetilación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Eliminación de Gen , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/genética , Sirtuinas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Elongación de la Transcripción Genética , Animales , Cromatina/química , Proteínas Cromosómicas no Histona/fisiología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/fisiología , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Ratones , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN no Traducido/biosíntesis , Sitio de Iniciación de la Transcripción , Transcripción Genética , Factores de Elongación Transcripcional/fisiologíaRESUMEN
Monomethylation of histone H3 at lysine 4 (H3K4me1) and acetylation of histone H3 at lysine 27 (H3K27ac) are correlated with transcriptionally engaged enhancer elements, but the functional impact of these modifications on enhancer activity is not well understood. Here we used CRISPR/Cas9 genome editing to separate catalytic activity-dependent and independent functions of Mll3 (Kmt2c) and Mll4 (Kmt2d, Mll2), the major enhancer H3K4 monomethyltransferases. Loss of H3K4me1 from enhancers in Mll3/4 catalytically deficient cells causes partial reduction of H3K27ac, but has surprisingly minor effects on transcription from either enhancers or promoters. In contrast, loss of Mll3/4 proteins leads to strong depletion of enhancer Pol II occupancy and eRNA synthesis, concomitant with downregulation of target genes. Interestingly, downregulated genes exhibit reduced polymerase levels in gene bodies, but not at promoters, suggestive of pause-release defects. Altogether, our results suggest that enhancer H3K4me1 provides only a minor contribution to the long-range coactivator function of Mll3/4.
Asunto(s)
Células Madre Embrionarias/enzimología , Elementos de Facilitación Genéticos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Regiones Promotoras Genéticas , ARN/biosíntesis , Transcripción Genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Edición Génica , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Masculino , Metilación , Ratones , Mutación , ARN/genética , Factores de Tiempo , Activación Transcripcional , TransfecciónRESUMEN
The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.
Asunto(s)
Células Madre Embrionarias/enzimología , Mamíferos/crecimiento & desarrollo , ARN Polimerasa III/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
By precisely tracking the waves of elongating RNA polymerase II (Pol II) during gene activation, Danko et al. (2013), in this issue of Molecular Cell, discovered a surprising diversity of elongation rates among and along human genes.
RESUMEN
Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.
Asunto(s)
Proteínas de Drosophila/genética , ARN Polimerasa II/fisiología , ARN Citoplasmático Pequeño/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Estabilidad del ARN , Transducción de Señal , Elongación de la Transcripción GenéticaRESUMEN
Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.
Asunto(s)
Empalme Alternativo , Animales Modificados Genéticamente/genética , Drosophila melanogaster/genética , Regulación Fúngica de la Expresión Génica , Poliadenilación , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Regiones no Traducidas 3'/genética , Animales , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , ARN Polimerasa II/genéticaRESUMEN
T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses.
Asunto(s)
Antígenos CD5/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , MicroARNs/genética , Poliadenilación , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Antígenos CD5/metabolismo , Regulación de la Expresión Génica , Humanos , Células Jurkat , Modelos Biológicos , Poli A , Interferencia de ARN , Isoformas de ARN , ARN Mensajero/genéticaRESUMEN
Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Poli A/metabolismo , Poliadenilación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Transducción de Señal/genética , Regiones no Traducidas 3'/genética , Animales , Animales Modificados Genéticamente , Proliferación Celular , Supervivencia Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Variación Genética/genética , Cinética , Poli A/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , ARN Polimerasa II/biosíntesis , ARN Polimerasa II/genéticaRESUMEN
RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.
Asunto(s)
Proteína Quinasa CDC2 , Carcinógenos , Melanoma , Estabilidad del ARN , ARN Nuclear , Neoplasias Cutáneas , Animales , Proteína Quinasa CDC2/genética , Melanoma/genética , Mutación , ARN Nuclear/genética , Neoplasias Cutáneas/genética , Pez Cebra , HumanosRESUMEN
The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.
Asunto(s)
ARN Helicasas DEAD-box/genética , Melanocitos/metabolismo , Cresta Neural/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Receptores de Progesterona/genética , Proteínas de Pez Cebra/genética , Animales , Línea Celular Tumoral , ARN Helicasas DEAD-box/metabolismo , Dihidroorotato Deshidrogenasa , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Humanos , Leflunamida/farmacología , Melanocitos/efectos de los fármacos , Melanocitos/patología , Cresta Neural/efectos de los fármacos , Cresta Neural/crecimiento & desarrollo , Nucleótidos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Unión Proteica , Receptores de Progesterona/metabolismo , Transducción de Señal , Estrés Fisiológico/genética , Elongación de la Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Alternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3' untranslated region (3'UTR), with critical physiological consequences. Here, we characterized the molecular mechanisms required for polo alternative polyadenylation. We identified a conserved upstream sequence element (USE) close to the polo proximal poly(A) signal. Transgenic flies without this sequence show incorrect selection of polo poly(A) signals with consequent downregulation of Polo expression levels and insufficient/defective activation of Polo kinetochore targets Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequence in vivo We found that Hephaestus binds to the USE RNA and that hephaestus mutants display defects in polo alternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of noncanonical polyadenylation signals in Drosophila melanogaster genes. Taken together, our results revealed the molecular mechanisms involved in polo alternative polyadenylation, with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a new in vivo function for USEs in Drosophila melanogaster.
Asunto(s)
Regiones no Traducidas 3' , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Animales , Secuencia de Bases , Secuencia Conservada , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Mitosis , Poliadenilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de SecuenciaRESUMEN
Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Nucleosomas/metabolismo , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción Genética , Animales , Factor de Unión a CCAAT/genética , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Células Madre Embrionarias , Técnicas de Silenciamiento del Gen , Ratones , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/metabolismoRESUMEN
The essential process of dosage compensation is required to equalize gene expression of X-chromosome genes between males (XY) and females (XX). In Drosophila, the conserved Male-specific lethal (MSL) histone acetyltransferase complex mediates dosage compensation by increasing transcript levels from genes on the single male X-chromosome approximately two-fold. Consistent with its increased levels of transcription, the male X-chromosome has enhanced chromatin accessibility, distinguishing it from the autosomes. Here, we demonstrate that the non-sex-specific CLAMP (Chromatin-linked adaptor for MSL proteins) zinc finger protein that recognizes GA-rich sequences genome-wide promotes the specialized chromatin environment on the male X-chromosome and can act over long genomic distances (~14 kb). Although MSL complex is required for increasing transcript levels of X-linked genes, it is not required for enhancing global male X-chromosome chromatin accessibility, and instead works cooperatively with CLAMP to facilitate an accessible chromatin configuration at its sites of highest occupancy. Furthermore, CLAMP regulates chromatin structure at strong MSL complex binding sites through promoting recruitment of the Nucleosome Remodeling Factor (NURF) complex. In contrast to the X-chromosome, CLAMP regulates chromatin and gene expression on autosomes through a distinct mechanism that does not involve NURF recruitment. Overall, our results support a model where synergy between a non-sex-specific transcription factor (CLAMP) and a sex-specific cofactor (MSL) creates a specialized chromatin domain on the male X-chromosome.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/fisiología , Compensación de Dosificación (Genética) , Proteínas de Drosophila/fisiología , Drosophila/genética , Cromosoma X , Animales , Genes Ligados a X , Historia Medieval , Masculino , Transcripción Genética/fisiologíaRESUMEN
Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning ('intron definition') or exon-spanning ('exon definition') pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60-70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. We observed unexpectedly low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.
Asunto(s)
Drosophila/genética , Precursores del ARN/metabolismo , Empalme del ARN , Animales , Modelos Teóricos , Análisis de Secuencia de ARNRESUMEN
Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a 'histone replacement' system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a post-transcriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression.