Mammalian cells acquire epigenetic hallmarks of human cancer during immortalization

Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK–ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK–ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.

consecutive positive probes, allowing one probe gap, covering a minimum length of 350 bp. This stringent definition will help preclude false-positive results. Identified methylation peaks were mapped to known transcripts defined in the UCSC Genome Browser MM9 RefSeq database (http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/). Methylation peaks falling into 1,000 bp relative to transcription start site (TSS) were defined as 'upstream' peaks; methylation peaks falling within 1,000 bp of RefSeq transcript end sites were defined as 'downstream' peaks, and those falling within gene bodies (from +1,000 bp of TSS to -1,000 bp of transcript end) were defined as 'intragenic' peaks. Methylation peaks that were not close to any known transcripts were defined as 'intergenic'. Methylation peaks in primary, senescent, and immortalized murine fibroblasts were identified as above. The methylated regions in immortalized samples were matched by outer join, and their methylation status was determined by the presence of methylation peaks in each sample in these matched regions. Only the regions methylated in the three immortalized cell lines and unmethylated in the respective primary fibroblasts were considered as hypermethylated candidates. The average log2 ratios of probes within these candidates in immortalized cells were compared to the average log2 ratios of probes in primary cells, and the final hypermethylation peaks were selected if the difference was more than 1 (over 2-fold). Conversely, hypomethylated candidates in immortalized cell lines were selected as those in which hypermethylated peaks were present in all of the primary fibroblasts with an average probe log2 ratio of >1 (over 2-fold) relative to immortalized cell lines. The annotated genes were analyzed using the DAVID: v 6.7 and the IPA: v 9.0 to provide gene enrichment analysis, functional annotation clustering, and details of the gene families found within the gene clusters.
We verified the methylation status of individual target loci/genes identified by MIRAmicroarray analysis using the conventional combined bisulfite restriction analysis (COBRA) (28), and sodium bisulfite sequencing (29). Briefly, one microgram of genomic DNA from primary, senescent or immortalized MEFs was subjected to sodium bisulfite treatment using the Qiagen EpiTect kit according to the manufacturer's instructions (Qiagen). The purified bisulfite-treated DNA was analyzed by standard COBRA assay (28) using sets of primers specifically designed for each target CpG island. The primer sequences used for PCR amplification of all the analyzed targets are shown in Table S1. Mouse genomic DNA was methylated with M. SssI CpG methyltransferase (New England Biolabs), and served as positive control. For genomic sequencing, the PCR products obtained after bisulfite conversion of genomic DNA were cloned into the TOPO-TA cloning vector according to the manufacturer's instructions (Invitrogen Inc.). Up to ten randomly selected clones from either primary, senescent or immortalized MEFs samples were sequenced using an ABI-3730 DNA Sequencer (ABI Prism, PE Applied BioSystems, Foster City, CA).
We have specifically determined the methylation status of major repetitive DNA elements, including long interspersed Single gene/locus DNA methylation analysis by COBRA and bisulfite sequencing.

DNA methylation analysis in repetitive DNA elements.
in vitro nuclear elements (LINE L1), interacisternal A particle long terminal repeat retrotransposons (IAP-LTR), and short interspersed nuclear elements (SINE B1) (30-32), in the genome of primary, senescent or immortalized MEFs using a bisulfite-based sequencing analysis (33). The method involves sodium-bisulfite treatment of the genomic DNA, followed by primer amplification of the consensus sequences from the respective elements, and subsequently direct DNA sequencing (33). Evolutionarily, methylated CpGs on the forward-or reverse strands of these elements can undergo spontaneous deamination of 5-methylcytosine to thymine, thereby, mutating to 5'-TpG-3' or 5'-CpA-3', respectively. Dependent on the status of cytosine methylation, non-mutated CpGs can be converted to 5'-TpG-3' (if unmethylated) or remain unchanged (if methylated) after bisulfite treatment of DNA (33). Thus, direct DNA sequencing of the bisulfitetreated and PCR-amplified fragments derived from these elements can provide detailed information on the status of CpG methylation and mutation in these elements (26,33).

Processing and analysis of Gene expression microarray data.
We confirmed the expression status of individual target genes identified by Affymetrix-microarray analysis using a standard quantitative realtime-PCR qRT-PCR) assay (34). Briefly, total RNA (0.5 g) from murine fibroblasts (before-crisis, crisis, and after-crisis) was reverse transcribed using the SuperScript® VILO™ cDNA Synthesis kit according to the manufacturer's instructions (Invitrogen).
The synthesized cDNA was then used for PCR amplification in the presence of the specific primer sets and the EXPRESS SYBR ® GreenER ™ qPCR SuperMix (Invitrogen).  Sodium-bisulfite sequencing of the CpG island in primary (1bc, 2bc, 3bc; left), senescent (1c, 2c, 3c; middle) and immortalized (1ac, 2ac, 3ac; right) MEFs. The sequencing results of several independent clones, and the respective percentage of methylation per sample are shown.  Hoxd10 Hoxd13 y Figure S5 Representative dot plots of differentially expressed genes in senescent and immortalized mouse fibroblasts counterpart primary cells versus