RESUMEN
Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.
Asunto(s)
Núcleo Celular , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromosomas/genética , Genoma Fúngico , Biología Sintética/métodosRESUMEN
Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Prosencéfalo/embriología , Oxidorreductasas de Alcohol/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Secuencia Conservada , Elementos de Facilitación Genéticos/genética , Proteínas de Homeodominio/fisiología , Interneuronas/fisiología , Ratones , Neurogénesis/genética , Neurogénesis/fisiología , ARN Largo no Codificante/genética , Factores de Transcripción , CohesinasRESUMEN
The Evf2 long non-coding RNA directs Dlx5/6 ultraconserved enhancer(UCE)-intrachromosomal interactions, regulating genes across a 27 Mb region on chromosome 6 in mouse developing forebrain. Here, we show that Evf2 long-range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2. Evf2 directly interacts with Sox2, antagonizing Sox2 activation of Dlx5/6UCE, and recruits Sox2 to the Dlx5/6eii shadow enhancer and key Dlx5/6UCE interaction sites. Sox2 directly interacts with Dlx1 and Smarca4, as part of the Evf2 ribonucleoprotein complex, forming spherical subnuclear domains (protein pools, PPs). Evf2 targets Sox2 PPs to one long-range repressed target gene (Rbm28), at the expense of another (Akr1b8). Evf2 and Sox2 shift Dlx5/6UCE interactions towards Rbm28, linking Evf2/Sox2 co-regulated topological control and gene repression. We propose a model that distinguishes Evf2 gene repression mechanisms at Rbm28 (Dlx5/6UCE position) and Akr1b8 (limited Sox2 availability). Genome-wide control of RNPs (Sox2, Dlx and Smarca4) shows that co-recruitment influences Sox2 DNA binding. Together, these data suggest that Evf2 organizes a Sox2 PP subnuclear domain and, through Sox2-RNP sequestration and recruitment, regulates chromosome 6 long-range UCE targeting and activity with genome-wide consequences.
Asunto(s)
Cromosomas de los Mamíferos/genética , Regulación del Desarrollo de la Expresión Génica , Prosencéfalo/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción SOXB1/genética , Animales , ADN Helicasas/genética , ADN Helicasas/metabolismo , Elementos de Facilitación Genéticos/genética , Técnica del Anticuerpo Fluorescente/métodos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación Fluorescente in Situ/métodos , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prosencéfalo/embriología , Unión Proteica , ARN Largo no Codificante/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The problem of three-dimensional (3D) chromosome structure inference from Hi-C data sets is important and challenging. While bulk Hi-C data sets contain contact information derived from millions of cells and can capture major structural features shared by the majority of cells in the sample, they do not provide information about local variability between cells. Single-cell Hi-C can overcome this problem, but contact matrices are generally very sparse, making structural inference more problematic. We have developed a Bayesian multiscale approach, named Structural Inference via Multiscale Bayesian Approach, to infer 3D structures of chromosomes from single-cell Hi-C while including the bulk Hi-C data and some regularization terms as a prior. We study the landscape of solutions for each single-cell Hi-C data set as a function of prior strength and demonstrate clustering of solutions using data from the same cell.