{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T13:07:43Z","timestamp":1792156063873,"version":"4.4.0"},"reference-count":33,"publisher":"Oxford University Press (OUP)","issue":"2","license":[{"start":{"date-parts":[[2022,5,9]],"date-time":"2022-05-09T00:00:00Z","timestamp":1652054400000},"content-version":"vor","delay-in-days":38,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["CBET-0967821"],"award-info":[{"award-number":["CBET-0967821"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,4,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>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 NF\u03baB 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 NF\u03baB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor\u2013DNA interactions. Antibodies to CREB1 and NF\u03baB 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 NF\u03baB 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 NF\u03baB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NF\u03baB consensus binding site was accompanied by more NF\u03baB recruitment to the promoter region.<\/jats:p>","DOI":"10.1093\/abt\/tbac010","type":"journal-article","created":{"date-parts":[[2022,5,9]],"date-time":"2022-05-09T12:46:45Z","timestamp":1652100405000},"page":"121-129","source":"Crossref","is-referenced-by-count":4,"title":["Evaluation of site-specific methylation of the CMV promoter and its role in CHO cell productivity of a recombinant monoclonal antibody"],"prefix":"10.1093","volume":"5","author":[{"given":"Hussain","family":"Dahodwala","sequence":"first","affiliation":[{"name":"National Institute for Innovation in Manufacturing Biopharmaceuticals , Newark, DE 19713 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7785-4336","authenticated-orcid":false,"given":"Sophia D","family":"Amenyah","sequence":"additional","affiliation":[{"name":"School of Biomedical Sciences, Ulster University , Coleraine, Co. Londonderry, BT52 1SA, Northern Ireland , UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Nicoletti","sequence":"additional","affiliation":[{"name":"College of Nanoscale Science and Engineering, SUNY Polytechnic Institute , Albany, NY 12203 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew N","family":"Henry","sequence":"additional","affiliation":[{"name":"Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland , St. Lucia, QLD 4072 , Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1309-1605","authenticated-orcid":false,"given":"Diane J","family":"Lees-Murdock","sequence":"additional","affiliation":[{"name":"School of Biomedical Sciences, Ulster University , Coleraine, Co. 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