Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation

Abstract

tIntraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenonthat has a strong impact on overall transgene expression levels and that limits the predictability of trans-gene expression in genetically modified cells, thereby hampering single cell based screening approaches.The underlying mechanism(s) leading to this variance are poorly understood. To study the dynam-ics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in morethan 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copyreporter cassettes driven by EF1alpha and CMV promoters. Single cell analysis showed intraclonal vari-ability with heterogeneity in expression in genetically uniform populations. DNA methylation is a wellknown mechanism responsible for silencing of gene expression. Interestingly, loss of expression wasnot associated with DNA methylation of the CMV promoter. However, in most of the clonal popula-tions expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting thatheterogeneity of transgene expression is crucially governed by histone modifications. Further, to deter-mine if the epigenetic status of transgene expression is governed by the chromosomal integration locuswe targeted heterologous expression cassettes into two chromosomal sites using recombinase medi-ated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-establishedwhen the same promoter used. In this way the problem of early stage cell clone instability can bebypassed. However, upon introduction of an unrelated promoter methylation-independent silencingwas observed. Together, these results suggest that histone modifications are the relevant mechanisms bywhich epigenetic modulation of transgene expression cassettes is governed in the early phase of clonegeneration.