Evaluation of site-specific methylation of the CMV promoter and its role in CHO cell productivity of a recombinant monoclonal antibody.

Autor:	Dahodwala H; National Institute for Innovation in Manufacturing Biopharmaceuticals, Newark, DE 19713, USA., Amenyah SD; School of Biomedical Sciences, Ulster University, Coleraine, Co. Londonderry, BT52 1SA, Northern Ireland, UK., Nicoletti S; College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY 12203, USA., Henry MN; Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD 4072 , Australia., Lees-Murdock DJ; School of Biomedical Sciences, Ulster University, Coleraine, Co. Londonderry, BT52 1SA, Northern Ireland, UK., Sharfstein ST; College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY 12203, USA.
Jazyk:	angličtina
Zdroj:	Antibody therapeutics [Antib Ther] 2022 May 06; Vol. 5 (2), pp. 121-129. Date of Electronic Publication: 2022 May 06 (Print Publication: 2022).
DOI:	10.1093/abt/tbac010
Abstrakt:	We previously demonstrated that increased monoclonal antibody productivity in dihydrofolate reductase (DHFR)-amplified CHO cells correlates with phosphorylated transcription factor-cytomegalovirus (CMV) promoter interactions. In this article, we extend the characterization to include CMV promoter methylation and its influence on NFκB and CREB1 transcription factor binding to the CMV promoter in two families of DHFR-amplified CHO cell lines. CMV promoter methylation was determined using bisulfite sequencing. To overcome Sanger-sequencing limitations due to high CG bias and multiple transgenes copies, pyrosequencing was used to determine the frequency of methylated cytosines in regions proximal to and containing the NFκB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor-DNA interactions. Antibodies to CREB1 and NFκB were used to immunoprecipitate formaldehyde-crosslinked protein-DNA fractions, followed by reverse transcription quantitative real-time polymerase chain reaction to quantitate the number of copies of CMV-promoter DNA bound to the various transcription factors. The relative unmethylated fraction at the CREB1 and NFκB consensus binding sites determined by pyrosequencing was correlated with transcription factor binding as determined by chromatin immunoprecipitation. Azacytidine treatment reduced methylation in all treated samples, though not at all methylation sites, while increasing transcription. Distinct promoter methylation patterns arise upon clonal selection in different families of cell lines. In both cell line families, increased methylation was observed upon amplification. In one family, the NFκB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NFκB consensus binding site was accompanied by more NFκB recruitment to the promoter region. (© The Author(s) 2022. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
Databáze:	MEDLINE
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