DNA-remethylation around a STAT5-binding enhancer in the αS1-casein promoter is associated with abrupt shutdown of αS1-casein synthesis during acute mastitis

Abstract

Prolactin stimulates the expression of milk genes during lactation through the activation of STAT5 transcription factors, which subsequently bind to their cognate target sequence on the promoters. Demethylation of 5methylCpG dinucleotides permits the tissue-specific accessibility of transcription factor-binding sites during development, but remethylation has not been shown to contribute to acute suppression of gene expression. We characterize functionally a novel STAT5-binding lactational enhancer in the far upstream promoter (wK10 kbp) of the bovine aS1-casein-encoding gene. This promoter area is hypomethylated in the lactating udder only. Remethylation of this area accompanies an experimentally elicited acute shutdown of casein synthesis in fully lactating cows, whose udder quarters have experimentally been infected with a pathogenic E. coli strain. Within 24 h after infection, the relevant promoter area was remethylated from 10% of the DNA molecules in the uninfected control quarters to w50% in the infected quarters, the typical values for fully lactating and not lactating udders respectively. Increased methylation resulted in tighter chromatin packing. Concomitantly, the aS1-casein mRNA concentration dropped to w50% while the protein synthesis was shut down to w2 . 5% in the infected quarters, alone. The methylation status of the promoter from a not lactationally regulated gene was unaltered, and the distal aS1-casein promoter was not remethylated in udder quarters with subclinical Staphylococcus aureus infections featuring sustained casein synthesis. Hence, infection-related remethylation of the aS1-casein promoter and chromatin remodelling serves as an acute, spatially restricted regulatory mechanism, which might insulate the promoter against the systemically unchanged high levels of circulating prolactin. This provides a rare example for an acute regulatory significance of CpG methylation.

Epigenetics: a possible role in acute and transgenerational regulation of dairy cow milk production

“…During Escherichia coli infection of the mammary gland, this region becomes methylated at three CpG dinucleotides and is associated with chromatin condensation. These changes accompany the shutdown of aS1-casein synthesis, with the mRNA levels dropping to 50% and protein levels to 2.5% of that in the non-mastitic control glands (Vanselow et al, 2006). Similar results have been demonstrated with a Streptococcus uberis infection of the mammary gland.…”

“…Demethylation may be passive or can also occur actively, although the process of demethylation mechanisms is less clear (Reik and Walter, 2001). Methylation may also occur at isolated CpG dinucleotides in close proximity to functional transcription factor binding sites (Vanselow et al, 2006). Furthermore, a small fraction of methylation also occurs in non-CpG cytosines (Ramsahoye et al, 2000).…”

“…DNA methylation of bovine aS1-casein gene during mastitis and involution Vanselow et al (2006) have described the association of DNA methylation and chromatin structure around a signal transducer and activator of transcription (STAT)5-binding lactation enhancer, which occurs at approximately 210 kb of the aS1-casein-encoding gene. In the bovine lactating mammary gland, this region of DNA is hypomethylated.…”

“…The methyl-CpG-binding proteins are thought to play a role in this interaction between DNA methylation and chromatin remodelling and modification (Klose and Bird, 2006). Epigenetic marks are generally maintained for the life of an organism; however, the acute nature of DNA methylation for regulating gene expression has been described (Vanselow et al, 2006;Kangaspeska et al, 2008;Mé tivier et al, 2008) and will be discussed below with respect to milk protein gene expression (see section 'Acute DNA methylation regulation of casein expression in mammary epithelial cells'). In mammals, there is limited evidence that these marks can be passed on to the next generation.…”

“…Hypomethylation during lactation has also been described for the bovine aS1-casein gene (Platenburg et al, 1996). Methylation and expression of this gene has been studied in mammary tissue of cows at different physiological states and during disease.DNA methylation of bovine aS1-casein gene during mastitis and involution Vanselow et al (2006) have described the association of DNA methylation and chromatin structure around a signal transducer and activator of transcription (STAT)5-binding lactation enhancer, which occurs at approximately 210 kb of the aS1-casein-encoding gene. In the bovine lactating mammary gland, this region of DNA is hypomethylated.…”