IRX5 promotes DNA damage repair and activation of hair follicle stem cells [nhek_d]

Here, we identify the transcription factor IRX5 as a promoter of HFSC activation. Irx5-/- mice display delayed onset of first postnatal anagen, with increased DNA damage and diminished HFSC proliferation. Through transcriptomic and epigenetic analysis, we discover the formation of open chromatin regions near key cell cycle progression- and DNA damage repair genes in Irx5-/- HFSC. We also identify DNA damage repair factors BRCA1 and BARD1 as IRX5 downstream targets. Inhibition of FGF18 kinase signaling partially rescues the anagen delay in Irx5-/- mice, indicating that the Irx5-/- HFSC quiescent phenotype is in part due to failure to suppress Fgf18 expression. Our findings identify IRX5 as a required promoter of DNA damage repair in HFSC activation and hair cycle initiation. Overall design: Normal Human Epidermal Keratinocytes (NHEK) were obtained from donor neonatal foreskin and grown in Keratinocyte Serum Free Media supplemented with Epidermal Growth Factor and Bovine Pituitary Extract (Life Technologies). Individual Dharmacon on-TARGETplus siRNAs [negative control (4390843), IRX2 (s45799, s45801), IRX3 (s35710, s35712), IRX4 (s55542, s27096), IRX5 (s20056, s20054)] were validated and siRNAs with at least 70% knockdown efficiency were pooled. 30nM pooled siRNAs were transfected into semi-confluent monolayers with Lipofectamine RNAi Max (Life Technologies) in OptiMEM medium. Twelve hours after transfection, 1.8mM Ca2+ was added into the growth medium to induce differentiation. RNA lysate was collected 72 hours after transfection.