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.
Highlights
Epigenetic modifications and regulations have attracted significant interest as researchers aim to identify the mechanisms that control gene expression
The coding gene pairs were extracted for these long non‐coding RNA (lncRNA) from our interaction list, and KEGG pathway enrichment was performed individually for coding genes corresponding to MALAT1 and NEAT1 homologues (Supporting Information Table 16)
Of the 885 genes reported by Capture Hybridization Analysis of RNA Targets (CHART)‐MS, 727 genes were found to be expressed in our cell lines, and 65% of those were identified in our triplex forming gene pairs corresponding to MALAT1 and NEAT1
Summary
Epigenetic modifications and regulations have attracted significant interest as researchers aim to identify the mechanisms that control gene expression. There has been a rapid increase in studies that describe transcriptional and posttranscriptional gene regulation by noncoding RNAs that function either independently or by interacting with other regulators (Dykes & Emanueli, 2017; Peschansky & Wahlestedt, 2014; Xu et al, 2017), and act as signals, decoys, guides, and scaffolds for chromatin modifiers (Marchese & Huarte, 2014; Wang & Chang, 2011) Together, these complex transcriptional dynamics result in defined patterns of gene expression and proteomic and metabolite profiles that determine the phenotype and cell survival. The resulting resource opens up possibilities both for enhanced control of cellular phenotypes during bioprocessing as well as the development of new engineering tools to manipulate cell behavior
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