First CpG island microarray for genome-wide analyses of DNA methylation in Chinese hamster ovary cells: new insights into the epigenetic answer to butyrate treatment

Abstract

Background: Optimizing productivity and growth of recombinant Chinese hamster ovary (CHO) cells requires insight and intervention in regulatory processes. This is to some extent accomplished by several 'omics' approaches. However, many questions remain unanswered and bioprocess development is therefore still partially empirical. In this regard, the analysis of DNA methylation as one of the earliest cellular regulatory levels is increasingly gaining importance. This epigenetic process is known to influence transcriptional events when it occurs at specific genomic regions with high CpG frequencies, called CpG islands (CGIs). Being methylated, CGIs attract proteins with methyl-DNA binding domains (MBD proteins) that in turn can interact with chromatin modifying complexes, thereby leading to a transcriptionally inactive state of the associated gene [1]. In CHO cells, DNA methylation has yet only been investigated in gene-specific approaches, e.g. regarding the CMV promoter [2]. To analyze differential DNA methylation in CHO cultures on a genomic scale, we developed a microarray covering 19,598 CGIs in the CHO genome. We applied it to elucidate the effect of butyrate on CHO DP-12 cultures, as this short chain fatty acid (SCFA) is known to elicit epigenetic responses by inhibiting histone-deacetylases [3]. Material(s) and Method(s): Based on the genomic and transcriptomic information available for CHO cells [4,5], 21,993 promoter-associated and intragenic CGIs were identified in the CHO genome using an algorithm according to Takai and Jones [6]. We developed a customized 60 K microarray (printed by Agilent Technologies) covering 19,598 (89%) of the identified CGIs with an average probe spacing of 500 bp. Genomic DNA of each four replicate experimental and reference CHO DP-12 (clone #1934, ATCC CRL-12445) batch cultures was phenol-chloroform extracted and sheared by sonication. Methylated fragments were enriched using the methyl-CpG binding domain of MBD2 protein fused to the Fc tail of IgG1 (MBD2-Fc protein) coupled to magnetic beads (New England Biolabs). Experimental samples prior to treatment with 3 mM butyrate (0 h) as well as 24 hours and 48 hours after butyrate addition were directly compared to the references by two-colour co-hybridizations. Data analysis was carried out upon LOWESS normalization by Student's t-tests with p-values