Epigenetic Modifications Control the Phenotype of CHO Cells Both during Short Term and Long Term Changes in Environment

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of recombinant therapeutic proteins as well as growth under a variety of culture conditions. The downside is that this adaptability comes with genomic and phenotypic instability. To understand the relative contribution of epigenetic regulation towards phenotype evolution, we analyzed full genome methylomes of 6 related cell lines that differ in adaptation to various culture conditions, in phenotype and in time in culture. The methylation pattern changed most significantly when cells were selected for a specific phenotype, such as sorting for increased productivity. Adaptation to new culture conditions or media composition resulted in intermediate changes in DNA methylation, while prolonged maintenance of cells under the same conditions has minimal impact. These results demonstrate that the behavior of production cell lines is determined by their gene expression pattern, which in turn controls their process relevant behavior and is itself controlled by epigenetics. Slight changes in culture environment such as observed during a batch or fedbatch culture can be expected to be likewise controlled by epigenetic mechanisms. For a more detailed understanding of the regulation underlying such changes, in addition to the methlyome, the histone modifications of a suspension adapted CHO cell line were analysed, along with the gene expression pattern at thight intervals throughout a batch culture. On top of CpG island methylation of promoters, the precise combination of histone marks at the promoter and TSS region controls the actual transcript level. Based on genome modifications, a model of chromatin states for CHO cells was calculated and it was shown that approx. 12 % of the genome change the chromatin state during a batch culture which results in clusters of genes that are up- or down-regulated upon transition from exponential growth to stationary phase, as well as gene clusters that are highly overexpressed or down-regulated only during a short period precisely at this transition. These modifications result in long term changes in the phenotype of cells, as demonstrated by the prolonged lag phase that is observed only if cells are maintained in culture long enough to undergo this transition. Epigenetic marks that are imprinted on cells during pre-cultures thus may have direct impact on the behavior of cells in a production run.