layer and then grown and expanded on 0.1% gelatin-coated plates (without feeder cells) in medium containing LIF for subsequent experiments. c-Myc Constitutive Knock-Out Mice The c-Myc constitutive knock-out mice were generated by crossing more-Cre and c-Myc floxed/floxed mice (a gift of I.M. de Albora´n) to delete the floxed c-Myc allele and then back-crossing to wild-type to remove more-Cre. The resulting c-Myc heterozy- gous mice were bred to obtain the c-Myc embryos for further experiments. Ethics Statement All research involving mice has been conducted according to the Institutional Animal Care and Use Committee Guidelines and has been pre-approved by the Institutional Review Board at the Fred Hutchinson Cancer Research Center. Chromatin Immunoprecipitation (ChIP) and DNA Microarrays (ChIP-chip) ES cells were purified by SSEA-1 flow sorting and treated with 1.1% formaldehyde for crosslinking which was terminated with 0.125M glycine before lysis and sonication to produce 0.2,1kb DNA fragments. Protein-A- agarose and protein-G-sepharose beads were blocked with 1 mg/ml sonicated salmon sperm DNA and 0.5 mg/ml BSA and washed twice in lysis/sonication buffer (50 mM Tris, 10 mM EDTA, 1%SDS, pH = 8). For ChIP with mouse monoclonal or rabbit polyclonal antibody, chromatin was pre-cleared with appropriate beads before immunoprecipitation overnight with antibody. Antibodies used for chromatin immuno- precipitation were c-Myc (Santa Cruz Biotechnology Inc. #SC764, Lot:F1307; Ab5 mouse monoclonal antibody from Thermo Scientific #MS-1054), H3K4me3 (Upstate, #05-745), and H3K27me3 (Upstate, #07-449), EZH2 (Abcam, #Ab3748- 100), and Suz12 (Upstate #07-379). The N262 antibody antibody has been used extensively for ChIP of c-Myc [27,31,81]. We further pre-absorbed the N262 antibody with electrophoretically fractionated proteins extracted from c-Myc embryos and demonstrated that the pre-absorbed antibody is specific for c-Myc protein and does not cross-react with N-Myc as determined by IP- western blotting. Chromatin immunoprecipitation and amplification (ligation- mediated PCR) for ChIP-chip analysis were carried out according to the Roche/NimbleGen protocol. Input DNA represents an aliquot of chromatin from the same sample used for ChIP and was crosslinked, sonicated, and processed identically to the ChIP sample. Duplicate samples were applied to the arrays. Our initial mouse promoter DNA microarray comprised 21,632 putative promoters from annotated mouse genes (NimbleGen, two mm8 promoter arrays). Using R software (http://www.R-project.org), we merged normalized data with the corresponding gene identifiers. Genes bound by Myc were identified as those in which any probe in the 5 kb promoter segment had a log2-ratio (ChIP vs. input DNA) .2. Data was further analyzed by the ACME package (www.bioconductor.org), a computer program developed for analysis of data obtained from NimbleGen-tiled microarrays, using a window of 500 bases and a threshold = 0.95 [58] [R Development Core Team (2008). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0]. To validate the ChIP-chip data from the promoter array, 1 ng input and ChIP DNA for Real-Time PCR was carried out using SYBR green PCR mix (Applied Biosystems) on ABI7900HT Myc Function in ES Cells detection system. The Ab5, c-Myc specific antibody was used for qChIP-PCR validation. The enrichment ratio was calculated by ChIP versus input and the positive targets were determined by criteria of 2-fold enrichment. We evaluated enrichment from duplicates of 2 independent ChIP assays obtained from both the R1 and AK7 ES cell lines. The sequences for all primers for ChIP- PCR are available upon request. Custom-design DNA microarrays for a subset of genes (see text) were tiled through .5 kb upstream and 1 kb downstream of transcription start sites using isothermal probes of 70–80 nucleotides. Triplicate samples were applied to the array. Genomic sites enriched for H3-K4me3 or H3-K27me3 binding were identified using ACME (window = 200, thresh = 0.9) and the given p values for each probes from ACME were plotted into graphs using the SAS 9.1 program. Analysis of Myc Target Genes To determine overlap among genes bound by c-Myc in mES cells (this paper) as well as by PcG and H3-K27me3 in mES cells we merged the list of genes on our arrays with those on the platform used by Boyer, et. al. [16].The majority of PcG bound sequences were detected within 1 kb of transcription start site [16]. Genes not included in both datasets were excluded from the analysis. Myc binding promoter –associated genes were annotated using Panther software (http://www.pantherdb.org/) [82]. Genes that are not assigned to a biological process (biological process unclassified) were not included in the figures. Each list is compared to the reference list (NCBI:M. Musculus genes) using the binomial test for each biological process term. Manipulating c-Myc Level in ES Cells by Lentiviral- Delivered Overexpression or shRNA Knock-Down c-Myc cDNA was amplified from ES-derived cDNA library and cloned into lentiviral vector pLenti6/V5-DEST (Invitrogen) for overexpression. The shRNA sequences against c-Myc was designed by using Dharmacon siDESIGN and then hairpin shRNA oligonucleotides were annealed and cloned into modified FUGW lenti-vector with H1 promoter (modified by Dr. Valera Vasioukhin). Lentiviral production was followed the protocol described in Lois C. et. al., Science, 2002, then drug selection for positive integration in ES cells followed by 3 days infection. Positive clones were picked and expanded for custom ChIP-chip array, qChIP-PCR, gene expression analysis by RT-PCR, and differentiation subsequent to LIF withdraw. Gene Expression Analysis by RT-PCR Total RNA was extracted by TRIzol (Invitrogen) reagent and cDNA was synthesized by SuperScript II kit (Invitrogen) according to the manufacturer’s instructions. The transcript expression level was measured by Real-Time PCR with SYBR Green detection on ABI7900HT system (Applied Biosystems). S16 RNA was used as internal control for normalization (DCt). The expression level was measured by subtraction of DCt from either overexpression or knock-down to vector control (DDCt). Primers for Real-Time PCR are available upon request. Proliferation and Growth Rate Assay FACS Analysis MTT assays: ES cells were seeded 1000 cells per well of 0.1% gelatin-coated 96-well plate supplied with LIF and analyzed 24, 48, 72, 96 hours after plating. At each time point, ES cell medium was replaced with 100 ml 1 mg/ml MTT (Molecular Probe) for labeling. Subsequently, 100 ml of SDS-HCl was added to each well and mixed thoroughly after 3 hours labeling at 37uC. Following 4 PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e7839