Ribotag RNA-seq of dermal fibroblasts from Tsc2 cKO mice identifies altered expression of genes that regulate hair cycle and mesenchymal cell differentiation

Summary: Abstract Body: Skin tumors in tuberous sclerosis complex (TSC) can display follicular changes associated with loss of TSC2 function in tumor fibroblasts and increased mTORC1 signaling. We developed conditional knockout mice (cKO), with Tsc2 deletion in mesenchymal cells and found increases in dermal thickness, hair shaft diameter, follicular density and hair regrowth. To understand the molecular mechanisms in vivo, Tsc2cKO or WT mice were mated with mice carrying a RiboTag allele. RNA sequencing on HA-immunoprecipitated RNA from mice leg skin was done to study changes in expression of translated genes in Tsc2-null or WT dermal fibroblasts (N=3 per group). Libraries were sequenced using the Illumina NextSeq platform. Reads were aligned to mouse transcripts using STAR and expression analysis was performed using DESeq2. We identified 927 differentially expressed genes, including 503 upregulated and 424 downregulated genes in leg skin dermal fibroblasts with Tsc2 deletion (p<0.05). In addition to the expected effect on the mTOR signaling pathway, Tsc2cKO also alters the expression of genes involved in hair cycle progression and hair growth, including upregulation of Fgf7, Gas6, Corin, Ctsl, as well as downregulation of Dkk1. Our data show that deep profiling of cell type-specific transcripts in vivo, accomplished by sequencing ribosome-associated transcripts, provides insights into molecular changes that may account for observed differences in skin structure and function. Ernest Phillips^{1, 2}, Sandhya Xavier^{1, 2}, Donald Aduba^{1, 2}, Peter Klover^{1, 2}, Rajesh Thangapazham^{1, 2}, Ji-an Wang^{1, 2}, Shaowei Li^{1, 2}, Jahnabi Roy^{1, 2}, Matthew Wilkerson^{3, 4}, Clifton Dalgard⁴, Joel Moss⁵, Thomas Darling¹ 1. Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States. 2. Henry M Jackson Foundation for the Advancement of Military Medicine Inc, Rockville, MD, United States. 3. Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD, United States. 4. The American Genome Center, Uniformed Services University of the Health Sciences, Bethesda, MD, United States. 5. Critical Care Medicine and Pulmonary Branch, National Heart Lung and Blood Institute, Bethesda, MD, United States. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics