Methods Mice: 4-6 weeks old female C57BL/6 mice obtained from Jackson labs were used for cell isolation. Female Foxp3-IRES-GFP reporter mice (1), backcrossed to B6/C57 background for 10 generations, were used for the isolation of naïve CD4 and naïve CD8 cells for the RNAseq experiments. The mice were housed in pathogen-free animal facility in the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee. Preparation of cells: Subsets of thymocytes were isolated by cell sorting as previously described (2), after cell surface staining using CD4 (GK1.5), CD8 (53-6.7), CD3ε (145- 2C11), CD24 (M1/69) (all from Biolegend). DP cells: CD4+CD8 int/hi; CD4 SP cells: CD4CD3 hi, CD24 int/lo; CD8 SP cells: CD8 int/hi CD4 CD3 hi, CD24 int/lo (Fig S2). Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4+CD62L hiCD25-CD44lo and naïve CD8+CD62L hiCD25-CD44lo were obtained by sorting (BD FACS Aria). Additionally, for the RNAseq experiments, CD4 and CD8 naïve cells were isolated by sorting T cells from the Foxp3- IRES-GFP mice: CD4+CD62LhiCD25–CD44lo GFP(FOXP3)– and CD8+CD62LhiCD25– CD44lo GFP(FOXP3)– (antibodies were from Biolegend). In some cases, naïve CD4 cells were cultured in vitro under Th1 or Th2 polarizing conditions (3, 4). Briefly, for Th1 polarization naïve CD4 T cells were cultured in the presence of 10 ng/ml IL12, 10 µg/ml anti-IL-4, 1 µg/ml anti CD3 (clone 2C11) and 1 µg/ml anti-CD28 (clone 37.51) for 2 days. In the case of Th2 polarization naïve CD4 T cells were cultured in the presence of 1000 U/ ml IL-4, 5 µg/ml anti-IFN-γ, 3 µg/ml anti-IL-12, 1 µg/ml anti CD3 (clone 2C11) and 1 µg/ml anti-CD28 (clone 37.51) for 2 days. On day 3 of culture, cells were removed from TCR/ costimulation conditions and expanded in the presence of 20 U/mL IL-2 for Th1 and 10 U/mL IL-2 for Th2 polarization conditions for 4 days. Data analysis was performed using FlowJo (Tree Star Inc). Intracellular staining for IFN-γ and IL-4: On day 6 of in vitro culture, cells were restimulated for 5 hours in the presence of 500 nM PMA (Calbiochem) and 1 µM ionomycin (Calbiochem). During the last 3 hours of culture, 10 µg/ ml of Brefeldin A (Sigma) was added. Intracellular staining was performed for IFN-γ and IL-4 using the Cytofix/Cytoperm kit (BD Biosciences). Data analysis was performed using FlowJo (Tree Star Inc). Enrichment-based detection of 5-hydroxymethylcytosine (5hmC): Biotin-glucosyl tagging and enrichment of 5hmC (Biotin-gmC, a variant of GLIB (5)): For specific biotinylation of 5hmC in DP T cells, we used the Hydroxymethyl Collector kit (Active Motif, Carlsbad, CA) to selectively label 5hmC bases with glucose and biotin moieties to form biotin-N3-5-gmC as previously described(6). To ensure efficient biotinylation, 2 µg of genomic DNA were biotinylated per reaction. In this case we used the 5500 SoliD fragment library preparation kit from Applied Biosystems in order to prepare the library and the 5500 SOLiD Library indexed adaptors 1-16 according to the manufacturer’s instructions. The samples were sequenced using the SOLiD 5500 platform. Chromatin Immunoprecipitation and library preparation: 10 million sorted DP T cells were fixed for 10 min in 1% PFA (Pierce), quenched with glycine and washed twice with PBS containing protease inhibitors (Roche) and sodium butyrate (to inhibit deacetylation) (Sigma). These inhibitors were present in all the following steps. Cells were lysed on ice in Lysis buffer (50 mM Hepes pH 7.9, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 140 mM NaCl, 10% Glycerol, 0.5% NP-40, 0.25 % Triton, protease inhibitors and 5 mM sodium butyrate) in order to obtain nuclei, that were washed twice with washing buffer (10 mM Tris-Cl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 1x protease inhibitors, 5 mM sodium butyrate) and resuspended in shearing buffer (0.1% SDS, 1 mM EDTA pH 8.0, 1x protease inhibitors, 5 mM sodium butyrate). Then, chromatin was sheared to an average size of 200 bp. Half of the material was used (equivalent to 5 million cells) per immunoprecipitation reaction. 10% was saved as input. Briefly, 2 µg of anti-histone H3acetylK27 (Abcam, ab 4729) was coupled to 30 µl Dynabeads protein G for immunoprecipitation (Invitrogen). The immunoprecipitation reaction was performed overnight (approximately 16 h) in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, 0.1% SDS, 0.5% Deoxycholate). The next day, 2 washes were performed with RIPA buffer, 1 wash with High Salt Buffer (50 mM Tris pH 8.0, 500 mM NaCl, 1mM EDTA, 1 % NP-40, 0.1% SDS, 0.5% Deoxycholate) and one wash with TE. After the last wash with TE, DNA was eluted by resuspending the beads in elution buffer (100 mM NaHCO3 and 1% SDS) with rotation for 40 min at room temperature. The eluted DNA from the IP reaction as well as the input DNA were treated with 5 ug of RNase A for 30 min to ensure removal of RNA. Then de-crosslinking reaction was performed overnight in the presence of proteinase K (Ambion) at 65 0C with shaking. De-crosslinked DNA was used to construct the library as described in the CMS-IP section, using the Illumina Truseq adaptors for the input and the IP sample. RNA isolation and whole transcriptome library preparation for RNAseq: For naïve CD4 and CD8 samples, RNA was isolated using Trizol (Invitrogen). Subsequently ribosomal RNA was depleted by RiboZero (Epicentre) and for the following steps the guidelines for whole transcriptome library preparation from low input provided by SOLiD (Applied Biosystems by Life Technologies) were followed. Briefly, using the total RNA- seq kit by SOLiD, enzymatic fragmentation of RNA was performed, then purification (PureLink RNA Micro Kit, Invitrogen), hybridization and ligation, reverse transcription, purification and size selection of the produced cDNA and amplification of the size selected DNA were performed using Phusion. The libraries were sequenced using the SoliD 5500 platform. Quantitative analysis of 5hmC levels using dot blots: 50 ng genomic DNA samples were sheared using needle and treated with sodium bisulfite using the Methylcode kit (Invitrogen), following the guidelines described in the CMS-IP section. DNA was prepared using 2-fold serial dilutions in TE buffer and then denatured in 0.4 M NaOH/ 10 mM EDTA at 95°C for 10 min and followed by adding an equal volume of cold 2M ammonium acetate (pH 7.0) on ice for 10 min. Denatured DNA samples were spotted on a nitrocellulose membrane in an assembled Bio-Dot apparatus (Bio-Rad) according to the manufacturer's instructions. The membrane was washed with 2XSSC buffer and then vacuum-baked at 80° C for 2 hrs. Then the membrane was blocked with 5% non-fat milk for 1 hr and incubated with rabbit antisera against CMS detected by HRP- conjugated secondary antibody and enhanced chemiluminescence. DATA ANALYSIS Biotin-gmC (GLIB) data analysis: The GLIB and input reads were mapped against mm9 in color-space using Bowtie (7). Enriched regions (ERGs) relative to input DNA were detected using the “findPeaks” routine in HOMER (8) with the “histone” mode and default parameter values. Intragenic levels of GLIB and input DNA were quantified by counting the number of reads falling into RefSeq genes defined by the corresponding annotated transcription start and termination sites. The RPKM values were calculated based on the read counts in the GLIB samples. The log fold enrichment of IP DNA was calculated against input DNA. CMS-IP data analysis: The CMS-IP and input reads were mapped against mm9 and lambda phage DNA using the Bismark mapping tool (9). The mapping was done using the Bowtie 2 (10) backend in the paired-end mode with the following parameter values: -I 0 -X 600 -N 0. Before pooling biological replicates their similarity was assessed using the method described below. Enriched regions (ERGs) relative to input DNA were detected using the “findPeaks” routine in HOMER (8) with the “histone” mode and default parameter values. Intragenic levels of CMS-IP and input DNA were quantified by counting the number of reads falling into RefSeq genes defined by the corresponding annotated transcription start and termination sites. The RPKM values were calculated based on the read counts in the CMS-IP samples. The log fold enrichment of IP DNA was calculated against input DNA. While calculating the average signal depicted in Fig. 1c-d the genes were extended 10% of the gene length downstream and upstream from transcription start site and transcription termination site, respectively. These extended gene regions were divided into 60 distinct intervals and the 5hmC and input DNA content of the bins was quantified (RPKM). For each of the bins the enrichment of 5hmC relative to input DNA was quantified followed by a step where the average enrichment was calculated over the considered genes. RNA-seq data analysis: The sequencing reads given either in base-space or color- space were mapped to the mm9 genome and exon-exon junctions using Tophat (11).