1. The upstream region of MUC5AC bound by SPDEF is required for the expression of MUC5AC in human lung cancer cells
The upstream region of MUC5AC bound by SPDEF is required for the expression of MUC5AC in human lung cancer cells Top panel, ChIP‐seq was performed twice using SPDEF antibody and chromatin from A549 cells as described in Materials and Methods. The SPDEF binding site was identified in the upstream region of the MUC5AC gene in A549 lung carcinoma cells (duplicate) and MCF7 breast adenocarcinoma cells (triplicate). Chr, chromosome. F (forward) and R (reverse) arrows indicate the primer locations to amplify the genome region that is bound by SPDEF. Bottom panel, two sgRNAs were designed to delete the SPDEF binding region.The non‐coding upstream region of MUC5AC that harbors different transcription factor‐binding sites. Red scissors indicate the locations where CRISPR/Cas9 made DNA double‐strand breaks.Three independent clones where precise deletion of the region (*) occurred. F (forward) and R (reverse) primers based on (A) were used to amplify the region that was bound by SPDEF.Only MUC5AC mRNA, but not other mucus‐related gene mRNAs, was significantly reduced in the three clones compared to the control (vector only) A549 cells. Gene expression was normalized by comparison with the constitutive expression of GAPDH. Results are expressed as mean ± SEM of biological replicates for each group. P < 0.05 versus control was considered significant (Student's t‐test). Untreated, no transfection (7 clones); control, vector only (eight clones); deleted, vector with sgRNAs (three clones).
Minzhe Guo et al. EMBO Mol Med. 2017;9:
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