RESUMO
Diverse DNA-deforming processes are impacted by the local mechanical and structural properties of DNA, which in turn depend on local sequence and epigenetic modifications. Deciphering this mechanical code (that is, this dependence) has been challenging due to the lack of high-throughput experimental methods. Here we present a comprehensive characterization of the mechanical code. Utilizing high-throughput measurements of DNA bendability via loop-seq, we quantitatively established how the occurrence and spatial distribution of dinucleotides, tetranucleotides and methylated CpG impact DNA bendability. We used our measurements to develop a physical model for the sequence and methylation dependence of DNA bendability. We validated the model by performing loop-seq on mouse genomic sequences around transcription start sites and CTCF-binding sites. We applied our model to test the predictions of all-atom molecular dynamics simulations and to demonstrate that sequence and epigenetic modifications can mechanically encode regulatory information in diverse contexts.
Assuntos
Fenômenos Biomecânicos , Metilação de DNA , Epigenoma , Animais , Camundongos , Ilhas de CpG/genética , DNA/química , DNA/metabolismo , Metilação de DNA/fisiologia , Sítio de Iniciação de Transcrição , Fenômenos Biomecânicos/fisiologiaRESUMO
C9ORF72 hexanucleotide GGGGCC repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-containing RNA mediates toxicity through nuclear granules and dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG translation. However, it remains unclear how the intron-localized repeats are exported and translated in the cytoplasm. We use single molecule imaging approach to examine the molecular identity and spatiotemporal dynamics of the repeat RNA. We demonstrate that the spliced intron with G-rich repeats is stabilized in a circular form due to defective lariat debranching. The spliced circular intron, instead of pre-mRNA, serves as the translation template. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron and modulates toxic DPR production. This study reveals an uncharacterized disease-causing RNA species mediated by repeat expansion and demonstrates the importance of RNA spatial localization to understand disease etiology.
Assuntos
Proteína C9orf72/genética , Núcleo Celular/metabolismo , Íntrons/genética , Biossíntese de Proteínas/genética , RNA/genética , Transporte Ativo do Núcleo Celular/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Expansão das Repetições de DNA/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
Almost all nucleoprotein interactions and DNA manipulation events involve mechanical deformations of DNA. Extraordinary progresses in single-molecule, structural, and computational methods have characterized the average mechanical properties of DNA, such as bendability and torsional rigidity, in high resolution. Further, the advent of sequencing technology has permitted measuring, in high-throughput, how such mechanical properties vary with sequence and epigenetic modifications along genomes. We review these recent technological advancements, and discuss how they have contributed to the emerging idea that variations in the mechanical properties of DNA play a fundamental role in regulating, genome-wide, diverse processes involved in chromatin organization.
Assuntos
Fenômenos Biomecânicos , DNA Super-Helicoidal/química , Genoma , Histonas/química , Nucleossomos/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Epigênese Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/metabolismo , Maleabilidade , Multimerização Proteica , Imagem Individual de Molécula , Torção MecânicaRESUMO
Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a 'mechanical code' with broad functional implications.