The Non-coding Mammary Carcinoma Susceptibility Locus, Mcs5c, Regulates Pappa Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation

In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman’s life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, Mcs5c, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that Mcs5c acts within the mammary gland to regulate expression of the neighboring gene Pappa during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. Mcs5c-mediated regulation of Pappa appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage.
Author Summary A woman’s lifetime risk of developing breast cancer is affected by both genetic and environmental risk factors that can be further exacerbated by breast developmental stage. Time periods conferring increased risk are referred to as Windows of Susceptibility (WOS) and, generally speaking, the molecular mechanisms responsible for their effect on breast cancer risk are unknown. Our work presented here on the characterization of the rat Mammary Carcinoma Susceptibility 5c, Mcs5c, locus has identified a region within Mcs5c that interacts with the neighboring gene, Pappa, in an age-dependent manner to influence gene expression via genotype-dependent DNA methylation. Importantly, Mcs5c-mediated gene regulation occurs specifically within a WOS, and these finding represent the first identified molecular mechanisms by which a WOS influences the ability of a locus to affect mammary/breast cancer risk. This work highlights the importance developmental stage can have on genetic risk factor function, and we anticipate that the Mcs5c locus will serve as a model for future studies on WOS in combination with genetic and environmental risk factors.