Comment[ArrayExpressAccession] E-GEOD-44036 MAGE-TAB Version 1.1 Public Release Date 2014-06-04 Investigation Title TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells [MeDIP-Seq] Comment[Submitted Name] TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells [MeDIP-Seq] Experiment Description We report that full length TET1 (TET1-FL) overexpression fails to induce global DNA demethylation in HEK293T cells. The preferential binding of TET1-FL to hypomethylated CpG islands (CGIs) through its CXXC domain leads to its inhibited 5-hydroxymethylcytosine (5hmC) production as methylation level increases. TET1-FL-induced 5hmC accumulates at CGI edges, while TET1 knockdown induces methylation spreading from methylated edges into hypomethylated CGIs. However, TET1 can regulate gene transcription independent of its dioxygenase catalytic function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically maintains the DNA hypomethylation state of CGIs in adult cells. hMeDIP-seq analysis of genomic 5-hydroxymethylcytosine in HEK293T cells overexpressing mTET1-CD, TET1-CD, mTET1-FL, or TET1-FL Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name JIN Jin Lu Jelinek Liang Estecio Barton Issa Person First Name CHUNLEI Chunlei Yue Jaroslav Shoudan Marcos Michelle Jean-Pierre Person Mid Initials R J Person Email jincl1980@gmail.com Person Affiliation The University of Texas MD Anderson Cancer Center Person Address The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, USA Person Roles submitter Protocol Name P-GSE44036-3 P-GSE44036-2 P-GSE44036-1 Protocol Description Sequenced DNA tags were mapped to human genome hg18 using ELAND (Illumina Analysis Pipeline) and uniquely mapped tags were kept. To avoid PCR bias, for the multiple tags that were mapped to the same genomic location, only one copy was kept. CCAT (version 3.0) was used to detect peaks in hMeDIP-Seq samples. The window size was set as 500 bp. Peaks with FDR M-bM-^IM-$ 0.05 and fold enrichment to input M-bM-^IM-% 5 were called significant. Genome_build: hg18 Supplementary_files_format_and_content: The columns of the processed file are: chr(chromosome of the peak), start(start postion of the peak), end(end position of the peak), tags_chip(number of tags in hMeDIP sample), tags_control(number of tags in Input sample), fold_change(fold change of hMeDIP to Input), FDR(false discovery rate). mTET1-CD, TET1-CD, mTET1-FL or TET1-FL expression plasmid (containing GFP reporter) was transfected into HEK293T cells by using OPTI-MEM (Invitrogen) and FuGene HD transfection reagent (Roche), followed by fluorescent activated cell sortng to collect GFP positive cells 3 (for mTET1-CD or TET1-CD ) or 7 (for mTET1-FL or TET1-FL ) days after transfection. The genomic DNA was then extracted from those cells. Before hydroxymethylated DNA immunoprecipitation (hMeDIP) reaction, genomic DNA samples were sonicated into a desirable fragment size of 100~500 bp. The purified sonicated DNA was then prepared for adapter ligation as per IlluminaM-bM-^@M-^Ys standard protocol (Illumina, San Diego, CA) with minor modifications. In brief, DNA was end-repaired using a combination of T4 DNA polymerase, Klenow enzyme (DNA Polymerase I Large Fragment) and T4 polynucleotide kinase (NEB, Ipswich, MA). The blunt phosphorylated DNA fragments were treated with Klenow fragment (3M-bM-^@M-^Y to 5M-bM-^@M-^Y exo minus) (NEB) and dATP to add an M-bM-^@M-^XAM-bM-^@M-^Y base to the 3M-bM-^@M-^Y end. Solexa paired ends adapters were ligated to the ends of the DNA fragments using Quick T4 DNA Ligase (Enzymatics, Beverly, MA). The resulting DNA products were subsequently subjected to hMeDIP for purifying 5hmC-containing DNA fragments. The immunoprecipitated and input control DNA were size selected by electrophoresis in a 2% agarose gel. A slice corresponding to 300 (M-1 25) bp size window based on DNA ladder was cut out. The DNA extracted from agarose was amplified with PCR (10-15 cycles) by using Solexa paired end PCR primers and PhusionM-bM-^DM-" High-Fidelity DNA Polymerase (NEB). The resulting sequencing library was finally cleaned with AMPure magnetic beads (Agencourt, Beverly, MA) and ready for single-read sequencing on Illumina Genome Analyzer II. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium modified with 10% fetal bovine serum and 100 M-NM-