Smad4 modulates expression of chemokine receptor Ccr6 in mouse colon stroma

Purpose: To understand how loss of epithelial Smad4 alters gene expression in mouse colon stroma Methods: To understand the extent of Smad-mediated gene regulation in the colon, we isolated colon epithelium from three Smad4?Lrig1 and from three 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 stroma by RNAseq. Results: A comparison of gene expression within the sub-epithelial stroma of Smad4?Lrig1 mice relative to SMAD4+ control mice demonstrated a marked increase in inflammatory gene signaling. Of the 95 genes that were upregulated by at least 2.5-fold (=1.32 Log2 Fold Change) and a false discovery rate <0.05, 51 genes are known to be expressed on immune cells or have immune-related functions, including Ccr6. Conclusions: SMAD4 modulates inflammatory signaling in colonic stroma to repress colitis-associated tumor formation Overall design: Three Smad4Lrig1 and three SMAD4+ control mice were dissected 1 month after administration of tamoxifen. Colon crypts were chelated using Ethylenediaminetetraacetic acid (EDTA). Following crypt chelation, the sub-epithelial stroma was removed from the underlying muscle layer by scraping. RNA was then made from the sub-epithelial stroma using RNeasy kit (Qiagen, Germantown, MD) and RNA-seq performed by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facility. 32–37 million 51–base pair, single-end reads were generated per sample. Reads were mapped to the mouse genome mm10 using TopHat-2.1.0, uniquely mapping 86%–95% single-end reads to the genome, depending on the study. The number of reads that fell into annotated genes were counted using samtools-1.3.1 and HTSeq-0.5.4p5. Count-based differential expression analysis was performed using edgeR_3.4.2.