Project description:We found that in addition to promoters, multiple Nups bind enhancers and insulators in the Drosophila genome. We identified a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Interestingly, although changing the levels of Nup98 disrupted induced enhancer-promoter contact, it did not affect transcriptional activation. Instead, loss of Nup98-mediated enhancer-promoter contact affected the primed response to subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with several architectural proteins upon stimulation with ecdysone.
Project description:Here we improved BiTS-ChIP (Bonn et al, Nature Protocols 7, 978-994 (2012)) to identify active enhancer and promoter elements genome wide in the 104 cardiomyocytes that constitute the Drosophila heart tube and represents only ~0.5% of the total cell content of the embryo. A transgenic Drosophila strain expressing nuclear GFP under the control of a cardiac specific enhancer (TinC*>GFP) was used for staged embryo collections at stages 13-14 (10-13h of development). After embryo fixation and dissociation, intact fixed nuclei were fluorescent labelling. Purification of this rare nuclear population was achieved by a two-step sorting procedure, yielding ~98% purity. Chromatin was extracted and used for immunoprecipitation and sequencing (ChIP-seq) to analyze chromatin modifications at promoters (H3K4me3 and H3K27ac) and enhancers (H3K27ac). Two independent biological replicates (from FACS sorting, chromatin preparations and ChIP-Seq) were performed for each mark and sequenced using Illumina HiSeq.
Project description:Here we improved BiTS-ChIP (Bonn et al, Nature Protocols 7, 978-994 (2012)) to identify active enhancer and promoter elements genome wide in the 104 cardiomyocytes that constitute the Drosophila heart tube and represents only ~0.5% of the total cell content of the embryo. A transgenic Drosophila strain expressing nuclear GFP under the control of a cardiac specific enhancer (TinC*>GFP) was used for staged embryo collections at stages 13-14 (10-13h of development). After embryo fixation and dissociation, intact fixed nuclei were fluorescent labelling.  Purification of this rare nuclear population was achieved by a two-step sorting procedure, yielding ~98% purity. Chromatin was extracted and used for immunoprecipitation and sequencing (ChIP-seq) to analyze chromatin modifications at promoters (H3K4me3 and H3K27ac) and enhancers (H3K27ac).  Two independent biological replicates (from FACS sorting, chromatin preparations and ChIP-Seq) were performed for each mark and sequenced using Illumina HiSeq.
Project description:What controls enhancer-promoter communication to allow for cis-transcriptional regulation remains a mystery. Here we studied how transcriptional dynamics shapes enhancer-promoter contacts. We demonstrate that enhancer function is reflected in enhancer-promoter contacts frequency which highly depend on active transcription. Pol II pausing, which is widespread across metazoan enhancers and promoters, has a direct effect on focal enhancer-promoter contacts. We confirmed this effect by depleting NELFB, a subunit of the negative elongation factor complex and a pivotal factor in Pol II pausing. Moreover, the catalytic activity of PARP1, a regulator of transcription and chromatin condensation, stabilizes enhancer-promoter contacts globally, but can destabilize contacts by promoting Pol II escape from pausing. Based on these findings, we propose an updated model that couples transcription and enhancer-promoter contacts.
Project description:What controls enhancer-promoter communication to allow for cis-transcriptional regulation remains a mystery. Here we studied how transcriptional dynamics shapes enhancer-promoter contacts. We demonstrate that enhancer function is reflected in enhancer-promoter contacts frequency which highly depend on active transcription. Pol II pausing, which is widespread across metazoan enhancers and promoters, has a direct effect on focal enhancer-promoter contacts. We confirmed this effect by depleting NELFB, a subunit of the negative elongation factor complex and a pivotal factor in Pol II pausing. Moreover, the catalytic activity of PARP1, a regulator of transcription and chromatin condensation, stabilizes enhancer-promoter contacts globally, but can destabilize contacts by promoting Pol II escape from pausing. Based on these findings, we propose an updated model that couples transcription and enhancer-promoter contacts.
Project description:What controls enhancer-promoter communication to allow for cis-transcriptional regulation remains a mystery. Here we studied how transcriptional dynamics shapes enhancer-promoter contacts. We demonstrate that enhancer function is reflected in enhancer-promoter contacts frequency which highly depend on active transcription. Pol II pausing, which is widespread across metazoan enhancers and promoters, has a direct effect on focal enhancer-promoter contacts. We confirmed this effect by depleting NELFB, a subunit of the negative elongation factor complex and a pivotal factor in Pol II pausing. Moreover, the catalytic activity of PARP1, a regulator of transcription and chromatin condensation, stabilizes enhancer-promoter contacts globally, but can destabilize contacts by promoting Pol II escape from pausing. Based on these findings, we propose an updated model that couples transcription and enhancer-promoter contacts.
Project description:Brain-derived Neurotrophic Factor (BDNF) promotes neuronal differentiation and survival and is implicated in the pathogenesis of many neurological disorders. Here, we identified a novel intergenic enhancer located 170 kb from the Bdnf gene, which promotes the expression of Bdnf transcript variants during mouse neuronal differentiation and activity. Following Bdnf activation, enhancer-promoter contacts increase, and the region moves away from the repressive nuclear periphery. Bdnf enhancer activity is necessary for neuronal clustering and dendritogenesis in vitro, and for cortical development in vivo. Our findings provide the first evidence of a regulatory mechanism whereby the activation of a distal enhancer promotes Bdnf expression during brain development.