Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

• Published in: Metabolic engineering Vol. 66; pp. 268 - 282
• Main Authors: Marx, Nicolas, Dhiman, Heena, Schmieder, Valerie, Freire, Catarina Martins, Nguyen, Ly Ngoc, Klanert, Gerald, Borth, Nicole
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.07.2021
• Subjects: CHO; CMV promoter; CRISPR/dCas9; DNA methylation; Epigenetic editing; Histone modifications; UHRF1; CRISPR/dCas9; H3K9me3; CHO; FACS; CMV promoter; ChIP; BFP; bp; gRNA; DNMT; FUT8; hCMV-MIE; HDR; LSD1; DNA methylation; KD; cDNA; sfGFP; CpG; ADCC; gDNA; GOI; PBS; CD; CRISPR; ST6GAL1; Epigenetic editing; H3K4me3; H3K27ac; CAG; dCas9; TET1; Histone modifications; KRAB; LCA; MSR; InDel; TSS; GlcNac; Cas9; NHEJ; MCS; BS-DNA; GAPDH; PAM; H3K27me3; HDAC
• DOI: 10.1016/j.ymben.2021.04.014
• ISSN: 1096-7176

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available. In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells. Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level. [Display omitted] •Endogenous and exogenous (viral) promoters respond differently to artificially induced modifications in their promoter methylation.•Cells autologously respond to altered promoter methylation by adapting the respective histone marks.•Promoters of genes that are expressed in normal CHO cells receive marks of facultative heterochromatin that can be easily re-activated.•Promoters of silent genes in CHO and the viral CMV promoter acquire histone marks of constitutive, permanent heterochromatin.