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SLAM-seq reveals early transcriptomic response mechanisms upon glutamine deprivation in Chinese hamster ovary cells
Maja Papež1, Víctor Jiménez Lancho1, Peter Eisenhut1
1Austrian Centre of Industrial Biotechnology (acib GmbH), Graz, Austria.
Abstract:
Mammalian cells frequently encounter subtle perturbations during recombinant protein production. Identifying the genetic factors that govern the cellular stress response can facilitate targeted genetic engineering to obtain production cell lines that demonstrate a higher stress tolerance. To simulate nutrient stress, Chinese hamster ovary (CHO) cells were transferred into a glutamine(Q)-free medium and transcriptional dynamics using thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) along with standard RNA-seq of stressed and unstressed cells were investigated. The SLAM-seq method allows differentiation between actively transcribed, nascent mRNA, and total (previously present) mRNA in the sample, adding an additional, time-resolved layer to classic RNA-sequencing. The cells tackle amino acid (AA) limitation by inducing the integrated stress response (ISR) signaling pathway, reflected in Atf4 overexpression in the early hours post Q deprivation, leading to subsequent activation of its targets, Asns, Atf3, Ddit3, Eif4ebp1, Gpt2, Herpud1, Slc7a1, Slc7a11, Slc38a2, Trib3, and Vegfa. The GCN2-eIF2α-ATF4 pathway is confirmed by a significant halt in transcription of translation-related genes at 24 h post Q deprivation. The downregulation of lipid synthesis indicates the inhibition of the mTOR pathway, further confirmed by overexpression of Sesn2. Furthermore, SLAM-seq detects short-lived transcription factors, such as Egr1, that would have been missed in standard experimental designs with RNA-seq. Our results describe the successful establishment of SLAM-seq in CHO cells and therefore facilitate its future use in other scenarios where dynamic transcriptome profiling in CHO cells is essential.
Insights
Chinese hamster ovary (CHO) cells under glutamine (Q) deprivation activate the integrated stress response (ISR) pathway. This study successfully established thiol-linked alkylation for metabolic sequencing of RNA (SLAM-seq) in CHO cells for dynamic transcriptome profiling.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biotechnology
Background:
- Mammalian cells, particularly Chinese hamster ovary (CHO) cells, face stress during recombinant protein production.
- Understanding cellular stress responses is crucial for engineering more robust production cell lines.
- Nutrient limitations, like glutamine (Q) deprivation, trigger significant cellular responses.
Purpose of the Study:
- To investigate the transcriptional dynamics of CHO cells under simulated nutrient stress (Q-free medium).
- To evaluate the utility of thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) for time-resolved transcriptome profiling in CHO cells.
- To identify genetic factors governing the cellular stress response for improved cell line engineering.
Main Methods:
- Simulated nutrient stress by transferring CHO cells to Q-free medium.
- Performed transcriptional profiling using standard RNA-sequencing (RNA-seq) and SLAM-seq.
- SLAM-seq enabled differentiation between nascent and total mRNA, providing time-resolved data.
Main Results:
- CHO cells activated the integrated stress response (ISR) pathway, evidenced by early Atf4 overexpression and subsequent target gene activation (Asns, Atf3, etc.).
- The GCN2-eIF2α-ATF4 pathway was confirmed by suppressed transcription of translation-related genes at 24 hours post-Q deprivation.
- Downregulation of lipid synthesis and Sesn2 overexpression indicated mTOR pathway inhibition. SLAM-seq detected short-lived factors like Egr1 missed by standard RNA-seq.
Conclusions:
- SLAM-seq is successfully established in CHO cells, offering a powerful tool for dynamic transcriptome analysis.
- The study elucidates the molecular response of CHO cells to amino acid limitation, involving ISR and mTOR pathway modulation.
- This methodology facilitates future research in CHO cell engineering for enhanced stress tolerance and improved bioproduction.
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