BioProject

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. More...

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. Overall design: Genomic DNA is isolated from 400K sorted native or formaldehyde fixed cells with Lysis Buffer (100 mM Tris-HCl pH 8.8, 5 mM EDTA, 200 mM NaCl, 0.2% SDS and 0.4 mg/ml Proteinase K) at 55 °C for 4 hours, sheared to the length of 300 – 500 bp and eluted in 20 µl of ddH2O. DNA methylation conversion is performed with EZ DNA Methylation-Gold kit (Zymo, #5005). Briefly, 20 µl of sheared genomic DNA is converted with 130 μl of the CT Conversion Reagent. The converted genomic DNA binds to Zymo-Spin™ IC Column and is proceeded for desulphonation with 200 μl of M-Desulphonation Buffer, and is eluted in 17 μl of Low EDTA TE Buffer. Bisulfite sequencing DNA libraries are prepared by using ACCEL-NGS® Methyl-seq DNA Library kit (Swift Biosciences, #30024 and #x6024) with 7 - 9 cycles’ PCR amplification as the manual described. Paired-end sequencing is performed in the Illumina NovaSeq 6000 system (2 × 150 bp) for WGBS. Less...