RESUMEN
Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.
Asunto(s)
Epigénesis Genética , Genoma Humano , Secuencias Repetitivas de Ácidos Nucleicos , Telómero/genética , Transcripción Genética , HumanosRESUMEN
The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR cis-regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention.
Asunto(s)
Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Transcripción Genética , Línea Celular Tumoral , Cromatina/química , Redes Reguladoras de Genes , Genoma Humano , Humanos , Masculino , Proteínas de Fusión Oncogénica/análisis , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/metabolismo , ARN Largo no Codificante/metabolismo , Regulador Transcripcional ERG/metabolismo , Regulador Transcripcional ERG/fisiologíaRESUMEN
The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.
Asunto(s)
Neoplasias Encefálicas/genética , Mapeo Cromosómico/métodos , Glioblastoma/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Cromatina/genética , Cromatina/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano , Glioblastoma/patología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células Jurkat , Desequilibrio de Ligamiento , Ratones , Ratones Desnudos , Elongación de la Transcripción GenéticaRESUMEN
The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulación de la Expresión Génica/genética , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular , ADN Polimerasa II/metabolismo , Proteínas de Unión al ADN/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Factores de Transcripción del Choque Térmico , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Factores de Transcripción/genéticaRESUMEN
We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.
Asunto(s)
Emparejamiento Base , Mapeo Cromosómico/métodos , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN/química , ARN/genética , Animales , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Humanos , Ratones , ARN/metabolismo , Análisis de Secuencia de ARN , Sitio de Iniciación de la TranscripciónRESUMEN
Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5'-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activationincluding expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor bindingthan those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function.
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Inteligencia Artificial , Regulación de la Expresión Génica/fisiología , Elementos Reguladores de la Transcripción/fisiología , Línea Celular , Estudio de Asociación del Genoma Completo , Histonas , Humanos , Células K562 , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Elementos Reguladores de la Transcripción/genética , Programas InformáticosRESUMEN
Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B cell (GM12878) and chronic myelogenous leukemic (K562) ENCODE Tier 1 cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses identify a common architecture of initiation, including tightly spaced (110 bp apart) divergent initiation, similar frequencies of core promoter sequence elements, highly positioned flanking nucleosomes and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modification and association of transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN/genética , Linfocitos B/citología , Sitios de Unión , Cromatina/química , Histonas/química , Humanos , Células K562 , Cadenas de Markov , Modelos Genéticos , Nucleosomas/química , Empalme del ARN , Secuencias Reguladoras de Ácidos Nucleicos , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.
Asunto(s)
Estabilidad del ARN/fisiología , ARN/clasificación , Exosomas/genética , Exosomas/fisiología , Células HeLa , Humanos , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN/fisiología , Estabilidad del ARN/genética , ARN no Traducido/fisiología , Iniciación de la Transcripción Genética/fisiología , Transcripción Genética/fisiologíaRESUMEN
Fluctuations in nutrient availability profoundly impact gene expression. Previous work revealed postrecruitment regulation of RNA polymerase II (Pol II) during starvation and recovery in Caenorhabditis elegans, suggesting that promoter-proximal pausing promotes rapid response to feeding. To test this hypothesis, we measured Pol II elongation genome wide by two complementary approaches and analyzed elongation in conjunction with Pol II binding and expression. We confirmed bona fide pausing during starvation and also discovered Pol II docking. Pausing occurs at active stress-response genes that become downregulated in response to feeding. In contrast, "docked" Pol II accumulates without initiating upstream of inactive growth genes that become rapidly upregulated upon feeding. Beyond differences in function and expression, these two sets of genes have different core promoter motifs, suggesting alternative transcriptional machinery. Our work suggests that growth and stress genes are both regulated postrecruitment during starvation but at initiation and elongation, respectively, coordinating gene expression with nutrient availability.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/genética , Genes de Helminto/genética , ARN Polimerasa II/metabolismo , Estrés Fisiológico/genética , Animales , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica , Mutación/genética , Regiones Promotoras Genéticas , Caperuzas de ARN/genética , ARN de Helminto/genética , ARN de Helminto/metabolismo , Análisis de Secuencia de ARN , Sitio de Iniciación de la Transcripción , Transcripción Genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs) in nascent RNA. Our results show that RDDs begin to occur in RNA chains ~55 nt from the RNA polymerase II (Pol II) active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.
Asunto(s)
ADN/metabolismo , ARN Polimerasa II/metabolismo , ARN/metabolismo , Dominio Catalítico , Técnicas de Cultivo de Célula , ADN/genética , Expresión Génica , Humanos , ARN/genética , Transcripción GenéticaRESUMEN
DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica , Variación Genética , Factores de Transcripción/metabolismo , Transcripción Genética , Alelos , Secuencia de Bases/genética , Sitios de Unión/genética , Cromatina/química , ADN/química , Histonas/química , Histonas/metabolismo , Humanos , Polimorfismo de Nucleótido Simple , Regiones Promotoras GenéticasRESUMEN
The X-chromosome gene regulatory process called dosage compensation ensures that males (1X) and females (2X) express equal levels of X-chromosome transcripts. The mechanism in Caenorhabditis elegans has been elusive due to improperly annotated transcription start sites (TSSs). Here we define TSSs and the distribution of transcriptionally engaged RNA polymerase II (Pol II) genome-wide in wild-type and dosage-compensation-defective animals to dissect this regulatory mechanism. Our TSS-mapping strategy integrates GRO-seq, which tracks nascent transcription, with a new derivative of this method, called GRO-cap, which recovers nascent RNAs with 5' caps prior to their removal by co-transcriptional processing. Our analyses reveal that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from the 5' ends of mature mRNAs. We find that C. elegans equalizes X-chromosome expression between the sexes, to a level equivalent to autosomes, by reducing Pol II recruitment to promoters of hermaphrodite X-linked genes using a chromosome-restructuring condensin complex. DOI:http://dx.doi.org/10.7554/eLife.00808.001.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas de Unión al ADN/fisiología , Compensación de Dosificación (Genética) , Complejos Multiproteicos/fisiología , Nematodos/genética , ARN Polimerasa II/metabolismo , Cromosoma X , Animales , Genoma , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Cascades of zygotic gene expression pattern the anterior-posterior (AP) and dorsal-ventral (DV) axes of the early Drosophila embryo. Here, we used the global run-on sequencing assay (GRO-seq) to map the genome-wide RNA polymerase distribution during early Drosophila embryogenesis, thus providing insights into how genes are regulated. We identify widespread promoter-proximal pausing yet show that the presence of paused polymerase does not necessarily equate to direct regulation through pause release to productive elongation. Our data reveal that a subset of early Zelda-activated genes is regulated at the level of polymerase recruitment, whereas other Zelda target and axis patterning genes are predominantly regulated through pause release. In contrast to other signaling pathways, we found that bone morphogenetic protein (BMP) target genes are collectively more highly paused than BMP pathway components and show that BMP target gene expression requires the pause-inducing negative elongation factor (NELF) complex. Our data also suggest that polymerase pausing allows plasticity in gene activation throughout embryogenesis, as transiently repressed and transcriptionally silenced genes maintain and lose promoter polymerases, respectively. Finally, we provide evidence that the major effect of pausing is on the levels, rather than timing, of transcription. These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints.
Asunto(s)
Tipificación del Cuerpo/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Proteínas de Drosophila/metabolismo , Femenino , Proteínas Nucleares , Factores de Transcripción/metabolismo , Cigoto/metabolismoRESUMEN
Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a precision nuclear run-on and sequencing (PRO-seq) assay to map the genome-wide distribution of transcriptionally engaged Pol II at base pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' polyadenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites, nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. This "complex interaction" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter.
Asunto(s)
Drosophila melanogaster/genética , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Iniciación de la Transcripción Genética , Transcripción Genética , Animales , Animales Modificados Genéticamente , Emparejamiento Base , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Genes de Insecto , Genoma de los Insectos , Proteínas HSP70 de Choque Térmico/genética , Modelos Genéticos , Mutagénesis Insercional , Nucleosomas/metabolismo , Sitios de Empalme de ARN , TransgenesRESUMEN
Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , ARN Polimerasa II/metabolismo , Animales , Inmunoprecipitación de Cromatina , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Polimerasa II/genética , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Fcp1 dephosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II) to recycle it into a form that can initiate a new round of transcription. Previously, we identified Drosophila Fcp1 as an important factor in optimal Hsp70 mRNA accumulation after heat shock. Here, we examine the role of Fcp1 in transcription of heat shock genes in vivo. We demonstrate that Fcp1 localizes to active sites of transcription including the induced Hsp70 gene. The reduced Hsp70 mRNA accumulation seen by RNA interference (RNAi) depletion of Fcp1 in S2 cells is a result of a loss of Pol II in the coding region of highly transcribed heat shock-induced genes: Hsp70, Hsp26, and Hsp83. Moreover, Fcp1 depletion dramatically increases phosphorylation of the non-chromatin-bound Pol II. Reexpression of either wild-type or catalytically dead versions of Fcp1 demonstrates that both the reduced Pol II levels on heat shock genes and the increased levels of phosphorylated free Pol II are dependent on the catalytic activity of Fcp1. Our results indicate that Fcp1 is required to maintain the pool of initiation-competent unphosphorylated Pol II, and this function is particularly important for the highly transcribed heat shock genes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Fosfoproteínas Fosfatasas/metabolismo , ARN Polimerasa II/metabolismo , Activación Transcripcional , Animales , Línea Celular , Drosophila/genética , Proteínas de Drosophila/genética , Eliminación de Gen , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico , Fosfoproteínas Fosfatasas/genética , Fosforilación , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Polimerasa II/química , ARN Mensajero/genéticaRESUMEN
Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0Fâ¼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0Fâ¼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Mapeo de Interacción de Proteínas , Secuencia de Aminoácidos , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Genes Reporteros , Viabilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética , Esporas Bacterianas/crecimiento & desarrollo , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Mutantes/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Embrión no Mamífero/enzimología , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Mutantes/genética , Mutación , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/genéticaRESUMEN
Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of â¼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Células Madre Embrionarias/enzimología , Regulación Enzimológica de la Expresión Génica , Animales , Diferenciación Celular , Células Madre Embrionarias/citología , Fibroblastos/citología , Fibroblastos/enzimología , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Ratones , Regiones Promotoras GenéticasRESUMEN
The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.