The methylation status of the nucleotide base, cytosine in DNA plays a major role in gene expression and transcriptional regulation, with methylation generally inhibiting gene expression. The CMV promoter, which is widely used to drive the expression of exogenous genes in mammalian cells, can be negatively affected by DNA methylation, due to its large number of CpG islands.  We hypothesized that the methylation/demethylation of this region in a collection of mAb producing CHO cell clones could explain our previous observation of increased mRNA per gene copy for MTX-amplified CHO cell clones.  To investigate this possibility, changes to the CMV promoter sequence as well as changes in transcription factor-promoter interactions have been studied.  In one study, several CHO cell clones were treated with Azacytidine, a DNA methyltransferase inhibitor known to activate gene expression. Azacytidine treatment increased specific productivity for some, but not all, low productivity clones and had limited effects on high productivity clones.  Genomic DNA from treated and untreated cells was analyzed by bisulfite sequencing to determine the effects of Azacytidine on the methylation status of the CMV promoter in the various clones. Insertional elements were observed in the CMV promoter in some of the clones as well as methylation differences between clones. To evaluate changes in promoter-DNA interactions, engineered transcription activator-like effectors (TALE) proteins were transiently transfected into the CHO cell clones.  These TALE proteins were designed to bind specifically to the CMV promoter, which could then be pulled down using chromatin immunoprecipitation along with their associated transcriptional proteins. <br/>

Epigenetic studies of antibody-producing CHO cells for therapeutic applications<br />

249th National Meeting (2015)

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