Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions −77 and − 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylationindependent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX's ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice. Keywords Cancer. H2AX. Mammary gland. Methylation. STAT5 Abbreviations DDR DNA-damage response GFP Green fluorescent protein Obs/Exp Observed to expected STAT Signal transducer and activator of transcription TET Ten-eleven translocation protein 5mC 5 methylcytosine 5hmC 5 hydroxymethylcytosine 5caC 5-carboxylcytosine 5fC 5-formylcytosine