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| Status |
Public on Sep 09, 2022 |
| Title |
DNA dioxygenases Tet2/3 control epithelial differentiation [CUT&TAG] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
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 CUT&Tag 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.
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| Overall design |
CUTANA™ CUT&Tag kit is purchased from Epicypher and the assay is carried out as the described manual. Briefly, nuclei of 100K sorted hair matrix keratinocytes are isolated with Nuclear Extraction Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1% TritonX-100, 20% Glycerol, 0.5 mM Spermidine and 1x cOmplete™ Protease Inhibitor Cocktail) and attached to Concanavalin A linked magnetic beads which are activated with Bead Activation Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2), and beads attaching nuclei are incubated with 0.5 µg of primary antibody in the Digitonin150 Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, and 1x cOmplete™ Protease Inhibitor Cocktail) containing 2 mM EDTA at 4 °C with gentle shaking for overnight and 0.5 µg of secondary antibodies in Digitonin150 Buffer at room temperature for 30 minutes successively. After washed twice with Digitonin150 Buffer, beads are incubated with 2.5 μl of pAG-Tn5 in Wash300 Buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, and 1x cOmplete™ Protease Inhibitor Cocktail) containing 0.01% Digitonin at room temperature for 1 hour. Beads attaching nuclei are washed with Wash300 Buffer containing Digitonin twice and incubated in Wash300 Buffer containing 10 mM MgCl2 at 37 °C for 1 hour to perform chromatin tagmentation. Beads are washed with TAPS Buffer (10 mM TAPS, pH 8.5 and 0.2 mM EDTA), and the DNA is released in 5 μl of SDS Release Buffer (10 mM TAPS, pH 8.5 and 0.1% SDS) at 55 °C for 1 hour and quenched with 15 μl of 0.67% Triton-X 100. DNA libraries are amplified for 18-21 cycles with 25 μl of NEBNext® High-Fidelity 2x PCR Master Mix (NEB, #M0541) and 2.5 μl of unique i5 and i7 index primers for Illumina. Paired-end sequencing is performed in Illumina NextSeq2000 P2 PE50 system (2 x 50bp). Antibodies: Dlx3 (Abcam, #ab66390), Lef1 (Cell Signaling, #2230), Pol2Ser5 (Abcam, #ab5408), CTCF (Diagenode, #C15410210), H3K4me1 (Diagenode, #C1541094), H3K4me3 (Abcam, #ab12209), H3K27ac (Abcam, #ab4729) and H3K27me3 (Millipore, #07-449).
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| Contributor(s) |
Chen G, Fatima I, Xu Q, Fessing MY, Mardaryev AN, Sharov AA, Rozhkova E, Xu G, Botchkarev VA |
| Citation(s) |
36630508 |
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| Submission date |
Sep 07, 2022 |
| Last update date |
Jan 24, 2023 |
| Contact name |
Guodong Chen |
| E-mail(s) |
[email protected]
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| Organization name |
Boston University
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| Department |
Dermatology
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| Street address |
609 Albany Street, ROOM 414
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| City |
BOSTON |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
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| Platforms (1) |
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| Samples (25)
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| This SubSeries is part of SuperSeries: |
| GSE212825 |
DNA dioxygenases Tet2/3 control epithelial differentiation |
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| Relations |
| BioProject |
PRJNA877587 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE212824_RAW.tar |
1.3 Gb |
(http)(custom) |
TAR (of BIGWIG) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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