DNA dioxygenases Tet2/3 control epithelial differentiation [RNA-seq]

Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes catalyzing 5-methylcytosine oxidation in DNA. Here, by using Keratin 14-Cre-driven ablation of Tet genes in skin epithelial cells, we demonstrate that ablation of Tet2/Tet3 result in marked alterations of hair shape and length followed by hair loss. We show that through DNA demethylation, Tet2/Tet3 control chromatin accessibility, Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. Moreover, Tet2/Tet3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via DNA methylation-independent mechanisms. These data demonstrate the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control. We performed RNA-seq from sorted hair matrix keratinocytes of wild type and TetDKO (Krt14-Cre, Tet2 flox/flox, Tet3 flox/flox) mice at P4.5 which are treated or untreated with 5-aza. Overall design: Hair matrix keratinocytes are isolated throgh FACS from mice at P4.5. For 5-aza treated mice, mice are treated with 5-aza at P2.5, and hair matrix keratinocytes are isolated following the same procedure decribed before. Total RNA is isolated from 100 - 200K sorted cells with RNeasy Micro Kit (QIAGEN, #74004) as the manual described, and eluted in 12 µl of DNase/RNase-free water. Zymo-Seq RiboFree Total RNA Library Kit (Zymo, #R3000) is used for cDNA synthesis and stranded library preparation as the manual described. DNA libraries are sequenced as the paired-end 2 × 150bp sequencing in an Illumina Hiseq4000 system.