B-G.45: A reproducible workflow for HEK293 methylome profiling during adaptation to suspension growth
Nikolaus Virgolini
BOKU - University of Natural Resources and Life Sciences Vienna
Georg Smesnik
BOKU - University of Natural Resources and Life Sciences Vienna
Astrid Duerauer
BOKU - University of Natural Resources and Life Sciences Vienna
Nicole Borth
BOKU - University of Natural Resources and Life Sciences Vienna
Keywords
DNA-Methylation Epigenetic HEK293
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Biomanufacturing for transient protein expression and recombinant Adeno-associated virus (rAAV) production often relies on empirical process development. Transitioning towards knowledge-driven optimization requires a fundamental understanding of host cell biology, which in the case of Human Embryonic Kidney 293 (HEK293) cells, the main expression system for rAAV production, is still limited. As such, this study presents a computational workflow for in-depth characterization of the epigenetic landscape of HEK293 cells and will be used in further investigations.
An adherent HEK293 cell line was adapted to suspension growth using four distinct commercial media formulations, with HEK293-6E serving as a commercially available suspension-adapted reference. Post-adaptation, DNA samples were taken and whole-methylome sequencing was performed using an enzymatic methyl-conversion kit and Illumina sequencing (150 bp paired-end, 37x coverage). The resulting data was evaluated, implementing a reproducible Snakemake-based processing and analysis pipeline. Raw data was processed and aligned to the human reference genome augmented with the Adenovirus5 region, prior to methylation calling via the Bismark program.
Next a downstream analysis pipeline was implemented, utilizing MethylKit and custom scripts to identify conserved methylomic signatures associated with suspension growth. To assess cell regulation, a chromatin state model was generated integrating publicly available datasets with ChromHMM. Mapping the experimental data against this model revealed characteristic mammalian patterns, specifically strong methylation within transcribed regions and lower levels at active transcription start sites and promoters. This work establishes a reproducible data analysis framework that provides critical insights into the HEK293 epigenome, offering a foundation for targeted host cell engineering.
Co-authors: Nikolaus Virgolini, Georg Smesnik, Astrid Duerauer, Nicole Borth
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