Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary (CHO) cell lines

Abstract

BackgroundRecombinant cell lines developed for therapeutic antibody production often suffer instability or lose recombinant protein expression during long-term culture. Heterogeneous gene expression among cell line subclones may result from epigenetic modifications of DNA or histones, the protein component of chromatin. We thus investigated in such cell lines, DNA methylation and the chromatin environment along the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter in an antibody protein-expression vector which was integrated into the Chinese hamster ovary (CHO) cell line genome.ResultsWe analyzed four PT1-CHO cell lines which exhibited losses of protein expression at advanced passage number (>P35) growing in adherent conditions and in culture medium with 10 % FCS. These cell lines exhibited different integration sites and transgene copy numbers as determined by fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR), respectively. By qRT-PCR, we analyzed the recombinant mRNA expression and correlated it with DNA methylation and with results from various approaches interrogating the chromatin landscape along the EEF1A1 promoter region. Each PT1-CHO cell line displayed specific epigenetic signatures or chromatin marks correlating with recombinant mRNA expression. The cell line with the lowest recombinant mRNA expression (PT1-1) was characterized by the highest nucleosome occupancy and displayed the lowest enrichment for histone marks associated with active transcription. In contrast, the cell line with the highest recombinant mRNA expression (PT1-55) exhibited the highest numbers of formaldehyde-assisted isolation of regulatory elements (FAIRE)-enriched regions, and was marked by enrichment for histone modifications H3K9ac and H3K9me3. Another cell line with the second highest recombinant mRNA transcription and the most stable protein expression (PT1-7) had the highest enrichments of the histone variants H3.3 and H2A.Z, and the histone modification H3K9ac. A further cell line (PT1-30) scored the highest enrichments for the bivalent marks H3K4me3 and H3K27me3. Finally, DNA methylation made a contribution, but only in the culture medium with reduced FCS or in a different expression vector.ConclusionsOur results suggest that the chromatin state along the EEF1A1 promoter region can help predict recombinant mRNA expression, and thus may assist in selecting desirable clones during cell line development for protein production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0238-0) contains supplementary material, which is available to authorized users.

CHO‐Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction

“…These results suggested a strong relationship between the epigenome and phenotype . Veith et al identified that during clonal selection cell lines display specific and unique epigenetic and chromatin signatures that correlate with expression . Feichtinger et al also tracked epigenetic changes through the course of adaptation, selection, passaging, and batch culture (Table ).…”

“…Second, methylation and histone patterns could be used as screening tools at early stages in the clone selection process. Epigenetic mechanisms have been identified to correlate with transgene expression profiles . Specific methylation and histone signatures could be identified to screen and enrich stable producers at an early time point in cell line development.…”

“…Using these types of models researchers were able to decipher how genomic or transcriptomic variation affected viability, metabolism, or protein expression for CHO . However, as the genomes and expression profiles of different CHO cell lines are highly variable, applying such methods to individual CHO lines would be very useful to determine cell line specific targets for genome engineering or medium development in order to maximize increases in product yield and quality. Iterating on these approaches, multiple CHO‐omic metabolic models were developed using murine metabolic models as a basis .…”

Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

“…In addition, CHO cells can be easily adapted to grow in suspension, in serum-free conditions and at high cell densities [10]. However, CHO cells possess also some unwanted traits, such as a relevant genome instability; they are also inclined to epigenetic silencing [11], [12]. Since undesired traits affect clone productivity (in terms of both quantity and quality), different strategies have been adopted to attenuate these disadvantages.…”

Fam60A plays a role for production stabilities of recombinant CHO cell lines

“…Histone deacetylation is among one of the cellular mechanisms that have been proposed to cause production instabilities (Veith et al, 2016). Assuming that due to the high amino acid sequence conservation of 97% between human and hamster FAM60A protein the function and interaction with HDACs is conserved, the knock-out of Fam60A or the deletion of Fam60A as in the CHO-C8DEL cell lines might increase production stabilities of cell lines through a lack of HDAC inhibition, leading to increased histone acetylation and therefore to open and active chromatin states.…”