DNA dioxygenases Tet2/3 control epithelial differentiation [ATAC-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 ATAC-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. ATAC-seq kit (active motif, #81286) is purchased from active motif company. As the manual described, 100K sorted cells are suspended in 100 μl ice-cold ATAC Lysis Buffer for nuclei extraction, and nuclei are incubated in 50 μl of Tagmentation Master Mix for DNA tagmentation. After purification, the DNA is amplified with NEBNext® High-Fidelity 2x PCR Master Mix (NEB, #M0541) as well as unique i5 and i7 index primers for 10 cycles to prepare DNA library. Paired-end sequencing is performed in the Illumina Nextseq500 Midoutput PE75 system (2 x 75 bp).