Long range regulation of transcription scales with genomic distance in a gene specific manner

ABSTRACT: While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we aim to bypass the need for sequence-specific transcription factors and recruit activators directly using catalytically dead Cas9 fused to the synthetic activator VPR. We perform a CRISPRa tiling screen in mouse ESCs that express doxycycline-inducible dCas9-VPR and were engineered with an endogenous T2A-EGFP reporter at the end of the Fgf5 gene. We used a library of over 13,000 guides spanning a 500kb window centered at the Prdm8-Fgf5 TAD. We demonstrate that this approach is unable to achieve detectable dCas9-VPR binding to arbitrary genomic sites making it impossible to address whether any genomic site to which activators are recruited can function as an enhancer. To overcome this limitation, we turned to CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.

ORGANISM(S): Mus musculus

PROVIDER: GSE278772 | GEO | 2024/12/02

REPOSITORIES: GEO