We have previously examined the regulatory region of the mouse lactoferrin gene and have identified sequences essential for basal and hormonally induced expression. In this study, we explore the relationship between the methylation state of the mouse lactoferrin gene promoter and its expression in selected mouse tissues. In a transient expression system, transcriptional activity was blocked after in vitro methylation of the regulatory region of the mouse lactoferrin gene. In addition, the in vivo methylation state of three promoter region sites was assessed using Southern blot analysis of DNA digested with methylation-insensitive and -sensitive restriction enzymes. The results showed that site -455, upstream of the mouse lactoferrin estrogen response module, was highly unmethylated in DNA from both hormone-treated and -untreated mouse lung, liver, and spleen tissues. Also, in both treated and untreated samples, the -54 site is uniquely highly unmethylated in liver DNA, while the -22 site is unmethylated in spleen DNA. Northern blot analysis showed lactoferrin expression in tissues that were unmethylated at a minimum of two sites. These results show that the alteration of the methylation status of the three sites are tissue-specific and are associated with constitutive expression of lactoferrin.

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