Smad4 pathways modulate induction of the chemokine Ccl20 and repress inflammation-induced carcinogenesis in mouse colon

To understand the extent of Smad-mediated gene regulation in the colon, we isolated colon epithelium from Smad4?Lrig1 and from Smad4+ control mice (either mice lacking a CreERT allele and treated with tamoxifen, or mice bearing a CreERT allele but treated with vehicle only) and analyzed the colonic epithelium by RNAseq. The ability of TGFß1 and/or BMP2 to block TNF-mediated induction of Ccl20 from our study suggests that these Smad-mediated pathways may act as gatekeepers for induction of other inflammation-associated genes. To determine if Smad-mediated signaling blocks all or specific subsets of TNF-induced genes, we analyzed both colonocytes and mouse colonoid treated with or without TNF, TGFß1, and BMP2 by RNA seq. Overall design: In total, three RNAseq experiments were performed and three biological replicates were used for each condition: 1. Colon epithelium from Smad4?Lrig1 and from Smad4+ control mice was isolated. Total RNA was isolated from these tissues using RNeasy kit (Qiagen). Processing of RNA using a TruSeq Stranded mRNA sample prep kit was conducted according to the manufacturer's instructions (Illumina, San Diego, CA). 32~37 million 51 base pair single-end reads were generated per sample. 2. Total RNA was isolated from colonocytes treated with or without TNF, TGFß1, and BMP2 using RNeasy kit (Qiagen). Processing of RNA using a TruSeq Stranded mRNA sample prep kit was conducted according to the manufacturer's instructions (Illumina, San Diego, CA). 26~50 million 75 base pair paired-end reads were generated per sample. 3. Total RNA was isolated from mouse colonoid treated with or without TNF, TGFß1, and BMP2 using RNeasy kit (Qiagen). Processing of RNA using a TruSeq Stranded mRNA sample prep kit was conducted according to the manufacturer's instructions (Illumina, San Diego, CA). 50~72 million 75 base pair paired-end reads were generated per sample.

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Means AL, Freeman TJ, Zhu J, Woodbury LG, Marincola-Smith P, Wu C, Meyer AR, Weaver CJ, Padmanabhan C, An H, Zi J, Wessinger BC, Chaturvedi R, Brown TD, Deane NG, Coffey RJ, Wilson KT, Smith JJ, Sawyers CL, Goldenring JR, Novitskiy SV, Washington MK, Shi C, Beauchamp RD