RNA-seq in Prdm16 KO duodenal crypts

The adult intestinal epithelium is maintained by a continuous replacement of differentiated cells from stem cells. Previous studies suggest that cellular metabolic pathways regulate intestinal stem cell activity and differentiation. However, little is known about the cell-intrinsic factors that control these metabolic programs. Here, we identify the transcription factor PRDM16 as a critical regulator of intestinal metabolic programing and stem cell differentiation. Acute deletion of Prdm16 in adult mice causes severe intestinal wasting, apoptosis, and an accumulation of poorly differentiated cells in the crypt. Prdm16-deficient crypts display decreased expression levels of fatty acid oxidation (FAO) genes and reduced rates of FAO. PRDM16 binds, along with its protein partners PPAR? and PPARa, to the promoter and enhancer regions of many FAO genes. The loss of Prdm16 or inhibition of FAO impaired the transition of intestinal stem cells into transit amplifying cells. Notably, PRDM16 expression is highest in the duodenum and declines distally along the intestine. This gradient of PRDM16 expression controls the region-specific expression of the FAO program and underlies the differential reliance of region-specific stem cells on FAO. Altogether, this study establishes PRDM16 as a regional-specific regulator of metabolism and stem cell differentiation in the intestine. Overall design: RNA was extracted from duodenal crypts isolated from crontrol and Prdm16 KO crypts 3 days after deletion RNA was isolated from duodenal crypts of WT (Prdm16loxP/loxP) or KO (Rosa26CreERT2; Prdm16loxP/loxP ) mice 3 days after tamoxifen injection. Total RNA was extracted from isolated crypts or enteroids (following one ice cold PBS wash to dissolve Matrigel) using TRIzol (Invitrogen) combined with Purelink RNA Mini columns (Fisher) and then reverse transcribed into cDNA using the ABI High-Capacity cDNA Synthesis kit. RNA-seq libraries were generated using a Truseq RNA Sample Preparation kit (Version 2, Illumina, RS-122-2001). RNA-seq was performed by the University of Pennsylvania Next Gen Sequencing Core using a HiSeq 2000 high output sequencer. Differential expression was determined using the EdgeR software package using log2 fold change of KO divided by WT. 0-1 False Discovery Rates were calculated from the p-value using a Benjamini-Hochberg correction.