Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems

Abstract

BackgroundThe DNA methylation profiles of mammalian cell lines differ from those of the primary tissues from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblasts to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs, suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0576-y) contains supplementary material, which is available to authorized users.

Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor α

“…Similarly, the Ewing sarcoma PDX models evaluated in this study were negative for PDGFC expression, despite previous work identifying PDGFC as a target gene of the EWS-FLI1 fusion transcription factor (42). These data are suggestive of potential alterations in the tumor transcriptome when cultured or propagated outside of its native environment and are reminiscent of a previous report describing changes in the epigenome and transcriptome of mammalian cells isolated from primary tissues upon exposure to cell culture (43). Furthermore, a recent study demonstrated that an anti-PDGFRa polyclonal antibody frequently used in previous IHC analyses cross-reacts with PDGFRb, further confounding previous reports regarding the prevalence of PDGFRa expression in pediatric bone and soft tissue tumor patient samples (44).…”

Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth

“…40 In addition to its role in DNA demethylation, 5hmC is a stable and functional epigenetic mark 41 and is enriched in the bodies of actively transcribed genes, at a number of transcription start sites, and at CTCF binding sites. 30,42,43 We identified significant enrichment of 5hmC at DMR2 and, although 5hmC is also known to be present at some imprinted DMRs, including across IGF2-H19, its precise role at these loci is unclear. 30 However, studies that show that H19 remains highly enriched for 5hmC in cell culture despite the global loss of 5hmC elsewhere, 43 and that such loci are also protected from demethylation in the post-fertilization embryo, 44 suggest that 5hmC dynamics at imprinted loci may differ from those in the remainder of the genome.…”

“…30,42,43 We identified significant enrichment of 5hmC at DMR2 and, although 5hmC is also known to be present at some imprinted DMRs, including across IGF2-H19, its precise role at these loci is unclear. 30 However, studies that show that H19 remains highly enriched for 5hmC in cell culture despite the global loss of 5hmC elsewhere, 43 and that such loci are also protected from demethylation in the post-fertilization embryo, 44 suggest that 5hmC dynamics at imprinted loci may differ from those in the remainder of the genome. Additionally, since 5hmC may be an intermediate in the DNA demethylation pathway, 5hmC at DMRs could result from the conversion of 5mC during the demethylation process.…”

H3K27me3 dynamics dictate evolving uterine states in pregnancy and parturition

“…Third, the genome-wide epigenetic changes that occur with adaptation to cell culture (e.g., see ref. 35) might preclude the generation of H3K27me3 marks in cultured DSCs.…”