Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture

Abstract

The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell‐line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9‐day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12‐ and 24‐hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription‐ and promoter‐specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non‐coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA–DNA–DNA triplexes. Such triplex‐forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex‐forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA‐triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes.

Bioinformatic Identification of Chinese Hamster Ovary (CHO) Cold‐Shock Genes and Biological Evidence of their Cold‐Inducible Promoters

“…A larger amount of differentially upregulated genes had a high fold change (483) than downregulated ones (73), showing that many genes increase transcription at 33 compared to 37 °C (Figure ). Three endogenous promoters P1‐RAD52 (short version, amplified by 1‐F4 and 1‐R14), P3‐ß actin, and P8‐HSP90 were generated in the previous study as constitutive endogenous promoters where their corresponding genes were not differentially expressed during the batch culture and TS experiment. Interestingly, these promoters also exhibited cold‐inducibility activity with up to 4.2‐fold increase in their promoter activities upon lowering the cultivation temperature in transient application (Figure 3A).…”

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

“…Potential promoter candidates were selected based on RNA‐Seq data of CHO‐K1 cells in the exponential growth phase including both coding genes and lnc transcripts . DNA regions upstream of highly expressed genes were visualized on the CHO epigenome browser to detect potential TSSs, promoter locations, and enhancer regions.…”

“…Promoter candidates were selected based on RNA‐Seq expression data . The CHO Epigenome Database was used to detect the locations of promoters and their enhancers based on chromatin states defined by histone marks.…”

“…In this study, selected CHO endogenous promoters and their enhancers were identified starting from RNA‐Seq and chromatin state data previously generated and tested for biological activity in comparison to viral promoters in transient transfections. Different CHO promoters were found ranging from low to very high activity compared to human cytomegalovirus promoter without/with enhancer (CMV P/P + E), which could enable precise control of gene expression for recombinant protein production and for systems biology engineering approaches in CHO cells.…”

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…Importantly, only 3% of the entire genome consists of protein coding genes, which are structured into exons and introns, while the remaining genome has a regulatory function or contains noncoding genes (Feichtinger et al, 2016;Hernandez et al, 2019;Hubé & Francastel, 2015). While the first human genome sequence became available in 2001, the CHO genome was first sequenced in 2011 only (Venter et al, 2001;Xu et al, 2011).…”