RESUMO
Mammalian gene expression is inherently stochastic1,2, and results in discrete bursts of RNA molecules that are synthesized from each allele3-7. Although transcription is known to be regulated by promoters and enhancers, it is unclear how cis-regulatory sequences encode transcriptional burst kinetics. Characterization of transcriptional bursting, including the burst size and frequency, has mainly relied on live-cell4,6,8 or single-molecule RNA fluorescence in situ hybridization3,5,8,9 recordings of selected loci. Here we determine transcriptome-wide burst frequencies and sizes for endogenous mouse and human genes using allele-sensitive single-cell RNA sequencing. We show that core promoter elements affect burst size and uncover synergistic effects between TATA and initiator elements, which were masked at mean expression levels. Notably, we provide transcriptome-wide evidence that enhancers control burst frequencies, and demonstrate that cell-type-specific gene expression is primarily shaped by changes in burst frequencies. Together, our data show that burst frequency is primarily encoded in enhancers and burst size in core promoters, and that allelic single-cell RNA sequencing is a powerful model for investigating transcriptional kinetics.
Assuntos
Genes/genética , Genômica , Transcrição Gênica/genética , Alelos , Animais , Elementos Facilitadores Genéticos/genética , Fibroblastos/metabolismo , Humanos , Cinética , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Especificidade de Órgãos/genética , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , Análise de Sequência de RNA , Deleção de Sequência , Análise de Célula Única , Processos Estocásticos , TATA Box/genética , Transcriptoma/genéticaRESUMO
This corrects the article DOI: 10.1038/cr.2018.1.
RESUMO
Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. Here, we show that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4) play an active role in this process. We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, our results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells.
Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Expressão Gênica/fisiologia , Hibridização in Situ Fluorescente , Metilação , Camundongos , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de RNA , CoesinasRESUMO
The pluripotency of embryonic stem cells (ESCs) is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although previous studies have identified transcriptional regulators of this core network, the cis-regulatory DNA sequences required for the transcription of these key pluripotency factors remain to be defined. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-enhancer (SE) located 100kb downstream of Sox2 in mouse ESCs. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy we deleted the entire 13-kb SE and characterized transcriptional defects in the resulting monoallelic and biallelic deletion clones with RNA-seq. We showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. Our results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Fatores de Transcrição SOXB1/genética , Animais , Linhagem Celular , Mapeamento Cromossômico , Biologia Computacional , Epigênese Genética , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Fatores de Transcrição SOXB1/metabolismo , Transcrição GênicaRESUMO
As the second dimension to the genome, the epigenome contains key information specific to every type of cells. Thousands of human epigenome maps have been produced in recent years thanks to rapid development of high throughput epigenome mapping technologies. In this review, we discuss the current epigenome mapping toolkit and utilities of epigenome maps. We focus particularly on mapping of DNA methylation, chromatin modification state, and chromatin structures, and emphasize the use of epigenome maps to delineate human gene regulatory sequences and developmental programs. We also provide a perspective on the progress of the epigenomics field and challenges ahead.
