Milking Frequency Modifies DNA Methylation at a CSN1 Regulatory Region in the Bovine Mammary Gland

Objectives: Milking frequency influences milk production in cows by regulating milk synthesis and secretion. This in turn involves a number of genes that are differentially expressed. In this study, we hypothesized that milking frequency has long term effects on milk yield through altered gene expression by modifying the methylation at CpG dinucleotides located within regulatory regions. In order to test this hypothesis on a candidate gene, DNA methylation of four CpG sites at the distal regulatory region of as1-casein (CSN1S1 - a major milk protein) was evaluated in relation to its gene expression.

Results: Global DNA methylation of the once daily milking group (75.9%) is higher compared to the twice daily milking group (74.6%) but only significant if one of the eight heifers, which behaved differently from the others, was excluded (P=0.04).

The DNA methylation of the three CpGs, which are more distal relative to the transcription start point, in the once daily milking group is higher than that of twice daily milking group (P<0.025, P~0.05, and P=0.025, respectively). This higher DNA methylation may explain the 2 fold lower CSN1S1 expression.

Conclusion: Once milking a day is related to a significant increase in DNA methylation at a distal CSN1S1 regulatory region and to a certain extent at the genomic global level in the bovine mammary gland. These modifications are in turn associated with modulated transcription of the casein gene and to some ncRNAs.