RESUMO
DNA-RNA hybrids are required for several natural processes in the cell, such as replication and transcription. However, the misregulation of its metabolism is an important source of genetic instability, a hallmark of diseases including cancer. For this reason, genome-wide detection of DNA-RNA hybrids is becoming essential to identify new factors that play a role in its formation or resolution and to understand the global changes in its dynamics because of genetic alterations or chemical treatments. Here, we describe two different immunoprecipitation-based procedures for the genome-wide profiling of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae: DRIP-seq and DRIPc-seq.
Assuntos
RNA , Saccharomyces cerevisiae , DNA/genética , Instabilidade Genômica , Humanos , Imunoprecipitação , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.
Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Gene order is not random in eukaryotic chromosomes, and co-regulated genes tend to be clustered. The mechanisms that determine co-regulation of large regions of the genome and its connection with chromatin three-dimensional (3D) organization are still unclear however. Here we have adapted a recently described method for identifying chromatin topologically associating domains (TADs) to identify coexpression domains (which we term "CODs"). Using human normal breast and breast cancer RNA-seq data, we have identified approximately 500 CODs. CODs in the normal and breast cancer genomes share similar characteristics but differ in their gene composition. COD genes have a greater tendency to be coexpressed with genes that reside in other CODs than with non-COD genes. Such inter-COD coexpression is maintained over large chromosomal distances in the normal genome but is partially lost in the cancer genome. Analyzing the relationship between CODs and chromatin 3D organization using Hi-C contact data, we find that CODs do not correspond to TADs. In fact, intra-TAD gene coexpression is the same as random for most chromosomes. However, the contact profile is similar between gene pairs that reside either in the same COD or in coexpressed CODs. These data indicate that co-regulated genes in the genome present similar patterns of contacts irrespective of the frequency of physical chromatin contacts between them.