Effects of CTCF on the regulatory landscape of the mouse Sox2 locus

Regulatory elements that control gene transcription are scattered along the genome, yet it is still poorly understood how their precise positioning affects the regulatory landscape. We previously reported a method to relocate a regulatory element to thousands of positions in a genomic locus and probe the position-dependent impact on gene regulation. Here, we apply this approach to systematically query the role and position-dependence of CTCF binding sites (CBSs), which are thought to modulate communication between distal regulatory elements. We focused on the mouse Sox2 locus in embryonic stem cells, where the Sox2 gene is activated by a potent distal enhancer. First, we found that a CBS that is naturally located immediately upstream of the Sox2 promoter endows this promoter with strong orientation-dependent activation, anywhere within the gene–enhancer interval. Second, throughout this interval, insertion of CBSs alone consistently reduces Sox2 expression in an orientation-dependent manner, suggesting a polarity in the loop extrusion process. Third, two native CBSs located within and downstream of the enhancer subtly help to confine the activation realm of the enhancer. Together, these results illuminate the interplay between CBSs and the detailed regulatory landscape of a genomic locus. This repository contains: PDF exports of all lab notebook entries related to the manuscript PDF export of the FACS sorting form the hopping experiments Plasmid maps for all used plasmids All gated and ungated fcs files used to generate the manuscript figures compressed into a zip file (each FACS file has a unique experiment number which refers back to the labnotes) Raw sequencing data is deposited to GEO (GSE324456), data anlysis pipelines and scripts to create all manuscrip figures are deposited in teh affiliated GitHub release on Zenodo. Note: use 7-zip to open the large zip-file with the lab notebook. Opening this zip-file in the windows file-explorer can give an error message.