Project description:Among epigenetic modifiers, telomeres, represent attractive modulators of the genome in part through position effects. Telomere Position Effect – Over Long Distances (TPE-OLD) modulates genes expression by changes in telomeric-dependent long-distance loops, with a reach of 10Mb from the telomeres. However, TPE-OLD remain poorly defined. We used cells with controlled telomere length combined to a genome wide approach revealing its transcriptome and methylome. We identified a common cis element that behaved as an insulator/ enhancer. By using reporter assays integrating this element, we disclosed additional trans partners further validated by in silico data. Exploiting our cellular model we observed the depletion of one candidate (RBPJ) at TPE-OLD associated loci concomitant to telomere shortening. Therefore, we conclude that TPE-OLD is orchestrated by Alu-like elements acting as enhancers in association with RBPJ. We propose that TPE-OLD functions by the coordinated action of newly evolved enhancers; an associated protein RBPJ and telomere length to produce an adapted response to external stimuli (i.e., Aging)
Project description:Among epigenetic modifiers, telomeres, represent attractive modulators of the genome in part through position effects. Telomere Position Effect – Over Long Distances (TPE-OLD) modulates genes expression by changes in telomeric-dependent long-distance loops, with a reach of 10Mb from the telomeres. However, TPE-OLD remain poorly defined. We used cells with controlled telomere length combined to a genome wide approach revealing its transcriptome and methylome. We identified a common cis element that behaved as an insulator/ enhancer. By using reporter assays integrating this element, we disclosed additional trans partners further validated by in silico data. Exploiting our cellular model we observed the depletion of one candidate (RBPJ) at TPE-OLD associated loci concomitant to telomere shortening. Therefore, we conclude that TPE-OLD is orchestrated by Alu-like elements acting as enhancers in association with RBPJ. We propose that TPE-OLD functions by the coordinated action of newly evolved enhancers; an associated protein RBPJ and telomere length to produce an adapted response to external stimuli (i.e., Aging)
Project description:The bacterial CRISPR-Cas9 (Cas9) nuclease has become a powerful genome manipulation tool for a wide range of organisms 1-3. However, it has yet to fully leverage the pervasive presence of DNA methylation in genomes4-10. To fill this gap, we report biochemical, structural, and human genome editing characterizations of a methylation-sensitive Cas9 (ThermoCas9). ThermoCas9 efficiently binds and cleaves DNA upstream of its protospacer adjacent motif (PAM) 5´-NNNNCGA-3´ or 5´-NNNNCCA-3´ in vitro. Methylation of the fifth cytosine in either PAM sequence (5mCpG or 5mCpC), however, significantly inhibits ThermoCas9 activity. Cryogenic electron microscopy structures of ThermoCas9 in pre- and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provides guidance for further enzyme engineering. We demonstrated methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrated that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in breast cancer patients. Due to its sensitivity to DNA methylation, ThermoCas9 can target cells specifically with disease-related hypomethylation which adds another layer of precision to genome editing technologies.