RESUMEN
Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.
Asunto(s)
Cromatina , Transcripción Genética , Regiones Promotoras Genéticas , Cromatina/genética , ARN sin Sentido/genética , Regulación de la Expresión GénicaRESUMEN
The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.
Asunto(s)
Proteínas de Drosophila , Elementos Aisladores , Animales , Ensamble y Desensamble de Cromatina , Nucleosomas/genética , Nucleosomas/metabolismo , Nucleasa Microcócica/genética , Nucleasa Microcócica/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Drosophila/genética , Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismoRESUMEN
CCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor implicated in a wide range of functions, including genome organization, transcription activation, and elongation. To explore the basis for CTCF functional diversity, we coupled an auxin-induced degron system with precision nuclear run-on. Unexpectedly, oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner independent of bound CTCF. Moreover, CTCF at different binding sites (CBSs) displays highly variable resistance to degradation. Motif sequence does not significantly predict degradation behavior, but location at chromatin boundaries and chromatin loop anchors, as well as co-occupancy with cohesin, are associated with delayed degradation. Single-molecule tracking experiments link chromatin residence time to CTCF degradation kinetics, which has ramifications regarding architectural CTCF functions. Our study highlights the heterogeneity of CBSs, uncovers properties specific to architecturally important CBSs, and provides insights into the basic processes of genome organization and transcription regulation.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Cromatina/metabolismo , Eritroblastos/metabolismo , Imagen Individual de Molécula , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Sistemas CRISPR-Cas , Línea Celular , Cromatina/genética , Ensamble y Desensamble de Cromatina , Edición Génica , Cinética , Ratones , Simulación de Dinámica Molecular , Unión Proteica , Proteolisis , ARN Polimerasa II/metabolismo , Transcripción GenéticaRESUMEN
The human gene catalogue is essentially complete, but we lack an equivalently vetted inventory of bona fide human enhancers. Hundreds of thousands of candidate enhancers have been nominated via biochemical annotations; however, only a handful of these have been validated and confidently linked to their target genes. Here we review emerging technologies for discovering, characterizing and validating human enhancers at scale. We furthermore propose a new framework for operationally defining enhancers that accommodates the heterogeneous and complementary results that are emerging from reporter assays, biochemical measurements and CRISPR screens.
Asunto(s)
Elementos de Facilitación Genéticos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma Humano , HumanosRESUMEN
Eukaryotic RNA polymerase II (Pol II) has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol II initiation, capping and pausing genome-wide. Our analyses identify two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, transcription start sites (TSSs) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is largely unchanged during the dramatic transcriptional changes induced by heat shock. Together, our results suggest that promoter- and enhancer-associated Pol II is a regulatory nexus for integrating information across TSS ensembles.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN/análisis , Análisis de Secuencia de ARN/métodos , Elongación de la Transcripción Genética , Transcripción Genética , Secuencia de Bases , Humanos , Células K562 , Unión Proteica , ARN/metabolismo , Caperuzas de ARN/metabolismo , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética/fisiología , Sitio de Iniciación de la TranscripciónRESUMEN
Because RNA-protein interactions have a central role in a wide array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay that couples sequencing on an Illumina GAIIx genome analyzer with the quantitative assessment of protein-RNA interactions. This assay is able to analyze interactions between one or possibly several proteins with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of the EGFP and negative elongation factor subunit E (NELF-E) proteins with their corresponding canonical and mutant RNA aptamers. Here we provide a detailed protocol for HiTS-RAP that can be completed in about a month (8 d hands-on time). This includes the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, HiTS and protein binding with a GAIIx instrument, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, quantitative analysis of RNA on a massively parallel array (RNA-MaP) and RNA Bind-n-Seq (RBNS), for quantitative analysis of RNA-protein interactions.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , ARN/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas/metabolismoRESUMEN
RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.