DOI: 10.5061/dryad.2z34tmq0n
title: CTCF couples long-range loop extrusion and diffusion to mediate a diverse Igk repertoire

description: Dynamic genome folding is important for V(D)J recombination at the
 immunoglobulin kappa (Igκ) locus, which recombines Jκ and Vκ gene segments
 across a 3.2 Mb region in both deletional and inversional orientations.
 Chromatin loop extrusion and diffusion are considered two key mechanisms
 underlying Igκ folding, but how they coordinate remains unclear.
 Here we show that CTCF is a key regulator coupling loop extrusion and
 diffusion during Igκ V-J rearrangement, promoting recombination in both
 orientations across long genomic distances. Mechanistically, the CTCF
 N-terminus promotes long-range loop extrusion that facilitates distal Vκ
 usage by stabilizing cohesin against WAPL release, and also forms loop
 barriers enabling chromatin diffusion for inversional Vκ joining. In CTCF
 N-terminal-deficient B cells, defects in inversional Vκ joining are not
 restored by WAPL depletion but are instead largely rescued by a
 dCas9-blockade targeted to the Vκ-Jκ intergenic region, mimicking the CTCF
 barrier. Our findings thus highlight how CTCF coordinates distinct
 genome-folding mechanisms through its dual roles in cohesin stabilization
 and extrusion barrier formation to ensure the generation of a
 diverse Igκ repertoire.
description: # CTCF couples long-range loop extrusion and diffusion to mediate a
 diverse Igk repertoire Dataset DOI:
 [10.5061/dryad.2z34tmq0n](https://doi.org/10.5061/dryad.2z34tmq0n) ##
 Description of the data and file structure This data was collected with
 the Attune Cytpix Flow Cytometer. In short, Rosa26-OsTIR1 and untreated
 and 1 uM 5-Ph-IAA treated Wapl-AID2 v-Abl pro-B cells were collected at
 indicated time points and cell cycle stages, spun down, PBS washed once,
 and resuspended in PBS with 2.5% FBS prior for flow cytometry analysis.
 ### Files and variables #### File: flow_cytometry_raw_data.zip
 **Description:** This .zip file contains the wild type and experimental
 .fcs files at the indicated time points during 5-Ph-IAA treatment and the
 subsequent G1 arrest of samples as previously described in the data
 description. Each file within the zip file is named according to the
 genotype, treatment, or non-treatment with 5-Ph-IAA, and timepoint of
 experiment, including the final G1 arrested timepoint. **WT_0hr.fcs** This
 is the parental, *Rosa26-OsTIR1(F74G) v-Abl* pro-B cell line collected at
 timepoint 0. **WT_NT_4hr.fcs** This is the
 parental, *Rosa26-OsTIR1(F74G) v-Abl* pro-B cell line collected at the
 4-hour timepoint, with no treatment with 5-Ph-IAA. **WT_NT_G1_day_4.fcs**
 This is the parental, *Rosa26-OsTIR1(F74G) v-Abl* pro-B cell line
 collected after being arrested with 3uM STI-571 for 4 days to induce G1
 arrest. **Wapl_AID2_NT_0hr.fcs** This is the *Wapl-AID2*  *v-Abl* pro-B
 cell line, collected at the time point 0, before treatment with 5-Ph-IAA.
 **Wapl_AID2_NT_4hr.fcs** This is the *Wapl-AID2* *v-Abl* pro-B cell line,
 collected at the 4-hour timepoint, with no treatment with 5-Ph-IAA.
 **Wapl_AID2_5-Ph-IAA_4hr.fcs** This is the *Wapl-AID2* *v-Abl* pro-B cell
 line, collected after treatment with 1 μM 5-Ph-IAA for 4 hours.
 **Wapl_AID2_NT_G1_day_4.fcs** This is the *Wapl-AID2* *v-Abl* pro-B cell
 line, after being arrested with 3uM STI-571 for 4 days to induce G1
 arrest, with no treatment with 5-Ph-IAA.
 **Wapl_AID2_5-Ph-IAA_G1_day_4.fcs** This is the *Wapl-AID2* *v-Abl* pro-B
 cell line, after being arrested with 3uM STI-571 for 4 days to induce G1
 arrest, while being continuously cultured with 1 uM 5-Ph-IAA. ## Software
 We used a licensed version of FlowJo v.10 to analyze our data. However,
 Floreada.io is a free, web-based application that can be used to view and
 analyze this dataset. Information about our gating strategies can be found
 in the source data associated with this manuscript.