Journal: Nature

Article Title: Loop Extrusion Mediates Physiological IgH Locus Contraction For RAG Scanning

doi: 10.1038/s41586-020-03121-7

Figure Lengend Snippet: a , Scatter plots of average transcriptome-wide GRO-seq counts in G1-arrested v-Abl cells ( x axis , n=3) and primary pro-B cells ( y axis , n=4). n , number of independent experiments. Representative known requisite genes implicated in the cohesin-complex function for V(D)J recombination and chromatin interactions are highlighted by red circles and blue arrows. Representative known genes implicated in the DNA repair and B cell development were also analyzed to determine if there were any potential transcriptional defects in these essential genes for V(D)J recombination and none were found and highlighted. Analyses of scatter plots indicate that Wapl is expressed at significantly higher levels in G1-arrested v-Abl cells than in primary pro-B cells (Spearman’s correlation coefficient (rho) and P values determined by two-sided Spearman’s correlation test are presented). These transcription finding were confirmed by western blotting studies . b , Comparison of Wapl transcription levels by GRO-seq in primary pro-B cells and G1-arrested v-Abl cells from 4 and 3 independent repeats, respectively. Data are presented as average signal counts ± s.e.m. of GRO-seq. n , number of independent experiments. Although other genes upstream and downstream of the Wapl gene show altered transcription in primary pro-B cells compared to the v-Abl cells, their products thus far have not been implicated in loop extrusion or chromatin scanning. c, Schematic of the targeting strategy for introducing Tet-On OsTIR1 expression cassette at the mouse Rosa26 locus. d, Strategy to generate Wapl-degron v-Abl cell lines. Positions of homology arms (gray box), Cas9/sgRNAs and southern blot probe are indicated. e, Southern blot confirmation of two correctly targeted clones (#5–3 and #5–9) with Wapl-mAID on both alleles (n=2 biologically independent repeats with similar results). f, Diagram of the experimental strategy to specifically deplete mAID-tagged Wapl protein in G1-arrested v-Abl cells. g, Western blotting to detect Wapl and Wapl-mAID protein. The indicated clones (#5–3 and #5–9) were grown without or with Wapl depletion at indicated time points before immunoblotting (n=2 biologically independent repeats with similar results). The specific western blotting bands of WT Wapl and Wapl-mAID were labeled. OsTIR1 was detected by anti-V5 antibody. Primary #5 clone was used for the WT Wapl control and β-actin was a loading control. h , Western blotting to detect Wapl protein levels in G1-arrested RAG1-deficient primary #5 v-Abl cells and cultured RAG1-deficient primary pro-B cells. Each sample was loaded with 3 indicated titrations. One of the two experiments is shown. i , Western blotting results to determine relative Wapl protein levels in cycling and G1 arrested Wapl-degron v-Abl cell clones (#5–3 and #5–9) with indicated conditions. For comparison, the intensity of the Wapl band in G1-arrested primary #5 v-Abl cells is set as 1.0. Average value is indicated at each bar. n , number of independent repeats. Indicated P values were calculated using unpaired two-tailed t -test. j, Representative flow-cytometry plots showing the percentage of Clover-positive Wapl-degron v-Abl cells that are without (Untreated) or with (IAA&Dox) Wapl depletion at indicated time points (n=2 biologically independent repeats with similar results). Primary #5 was processed as a Clover-negative control. k, Cell viability assay for G1-arrested v-Abl cells with indicated conditions. Data are presented as average percentage ± s.d. of viable cells for each condition. n , number of independent experiments. l, Representative flow-cytometry plots of propidium iodide (PI) stained G1-arrested v-Abl cells with indicated conditions (n=2 biologically independent repeats with similar results). Percentages in the top-right corner represent the percentage of cells at G1, S and G2/M stage. One of the two experiments is shown. Indicated P values were calculated using unpaired two-tailed t -test ( i , k ). Plasmids, sgRNAs and oligos used are listed in .

Article Snippet: The targeting plasmid (pTet-On_OsTIR1_V5) for introducing the doxycycline inducible OsTIR1-V5 expression cassette into endogenous Rosa26 locus was constructed by modifying a published pEN113 plasmid (Plasmid #86233, Addgene).

Techniques: Western Blot, Comparison, Expressing, Southern Blot, Clone Assay, Labeling, Control, Cell Culture, Two Tailed Test, Flow Cytometry, Negative Control, Viability Assay, Staining