Journal: bioRxiv

Article Title: Chromatin interaction maps identify Wnt responsive cis -regulatory elements coordinating Paupar-Pax6 expression in neuronal cells

doi: 10.1101/2021.05.18.442939

Figure Lengend Snippet: (A) N2A cells were transfected with pCl β-catenin S33Y or pCl empty vector and harvested for expression analysis 2 days later. Results are presented relative to the pCl control. (B, C) N2A cells were treated with either 50 ng/μl Wnt3a (B) or 0.1 ng/μl BMP4 (C) and harvested for RT-qPCR analysis 3 days later. 0.02% BSA in PBS was used as a negative control. (D) Genome browser graphic showing the ChIP amplified candidate CREs as well as the location of the TCF7L2 motifs and sgRNAs used in the CRISPRi experiment (GRCm38/mm10). (E) ChIP assays were performed in N2A cells using either an antibody against TCF7L2 or an isotype specific control. TCF7L2 occupancy at the indicated CREs was analysed by qPCR. Fold enrichment was calculated as 2 −ΔΔCt (IP/IgG) and is presented as mean value +/− sem. n≥3. One-tailed student’s t-test * p<0.05. (F) TCF7L2 levels were knocked-down in N2A cells by transfection of an endoribonuclease-prepared pool of TCF7L2 esiRNAs. An esiRNA pool targeting the luciferase gene was used as a negative control. Cells were harvested for expression analysis 3 days after transfection. (G) N2A cells were transfected with a plasmid co-expressing dCas9-KRAB and a sgRNA targeting the TCF7L2 motif within the indicated candidate CREs. A non-targeting sgRNA was used as a control. For all RT-qPCR reactions: Tcf7l2, Paupar and Pax6 expression levels were measured using RT-qPCR and results were normalised to Tbp. Results are presented as mean values +/− sem, n≥3. One-tailed student’s t-test * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: For Wnt and Bmp treatment, approximately 3 x 10 5 N2A cells were seeded per well in a 6-well plate in either growth medium containing 50 ng/ μl Wnt3a (R&D Biosystems, 5036-wn) or in low serum medium (DMEM supplemented with 5% FBS) containing 0.1 ng/ μl BMP4 (Thermo Fisher, PHC9534) as described in (Szemes, Melegh et al. 2020).

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Negative Control, Amplification, One-tailed Test, esiRNA, Luciferase