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Unbiased screening of regulatory insulators

ABSTRACT: Unbiased screening of regulatory insulators

PROVIDER: PRJNA1064098 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27505601_1.fastq.gz	Fastqsanger.gz
	SRR27505601_2.fastq.gz	Fastqsanger.gz
	SRR27505602_1.fastq.gz	Fastqsanger.gz
	SRR27505602_2.fastq.gz	Fastqsanger.gz
	SRR27564114_1.fastq.gz	Fastqsanger.gz

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Unbiased screening of regulatory insulators

Project description:Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes raises the need for mechanisms to limit inappropriate gene regulation. DNA elements known as insulators provide one such mechanism by shielding promoters from an enhancer when placed in between these two elements. Whereas promoters and enhancers have been extensively characterized in animal genomes, insulators have not because of the lack of a high-throughput screening assay. In the absence of unbiased insulator screening in a genome, basic questions remain about the identity, density and diversity of genomic insulators. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila tissue culture cells to perform the first unbiased screen for insulators in any genome. By screening developmental gene loci, we found that not all characterized insulator protein binding sites are able to block enhancer-promoter communication. By dissecting the sequence determinants of functional insulators, we found that an unexpectedly broad sequence context around the central insulator protein binding motif is required for functionality. The ability to screen millions of DNA sequences in parallel without any positional effect has enabled the first functional mapping of insulators and provided further insights into the determinants of functional insulators.

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Project description:The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs), within which the majority of gene-enhancer interactions occur. Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries3,4. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, mitotically-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing with dCas9-KRAB and dCas9-DNMT3A3L. Finally, we apply these strategies to activate PDGFRA expression in glioblastoma stem cells, thus simulating an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

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Project description:The many modifications involved in transcription are extensively coupled, and levels of histone modifications, insulators, and RNA polymerase II could be measured using genomic tiling microarrays. After 0, 30, 60 min of stimulation of HUVEC with TNF alpha, we crosslinked cells and fixed, then immunoprecipitated to prepare DNA using antibodies for modified histone, insulators and RNA polymerase. We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course

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