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

Anna Wippermann,Oliver Rupp,Sandra Klausing,Thomas Noll,Raimund Hoffrogge

doi:10.1186/1753-6561-7-s6-o5

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].

Highlights

Optimizing productivity and growth of recombinant Chinese hamster ovary (CHO) cells requires insight and intervention in regulatory processes
Gene ontology classification showed that, amongst others, the terms ‘stress response’, ‘chromatin modification’ or ‘signalling cascade’ were significantly overrepresented. Pathways such as the Ca2+, MAPK and Wnt signalling systems were comprised within the latter group and showed a large coverage by differentially methylated components. 48 hours upon butyrate addition the number of differential methylations decreased by about 90 %
Our first genome-wide screening for differential DNA methylation in CHO cells shows that the epigenetic response upon butyrate treatment seems to be highly dynamic and reversible

Summary

Introduction

Optimizing productivity and growth of recombinant Chinese hamster ovary (CHO) cells requires insight and intervention in regulatory processes. 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. Confirmatory COBRA (combined bisulfite restriction analysis) was performed by amplifying a 541 bp fragment of the myc proto-oncogene protein-like gene (Gene ID: 100758352) following bisulfite treatment of genomic DNA using the primers myc_for 5’-atttggaaggatagtaagtatattggaag-3’ and myc_rev 5’- aaataaaactctaactcaccatatctcct-3’ and the nested primers myc_for_nested 5’- atagtaagtatattggaaggggagtg-3’ and myc_rev_nested 5’- taaaactctaactcaccatatctcctc-3’ (oligonucleotides obtained from Metabion).

Results

Conclusion