Pregnancy causes a series of cellular and molecular changes in mammary epithelial cells (MECs) of female adults. In addition, pregnancy can also modify the predisposition of rodent and human MECs to initiate oncogenesis. Here, we investigate how pregnancy reprograms enhancer chromatin in the mammary epithelium of mice and influences the transcriptional output of the oncogenic transcription factor cMYC. We find that pregnancy induces an expansion of the active cis-regulatory landscape of MECs, which influences the activation of pregnancy-related programs during re-exposure to pregnancy hormones in vivo and in vitro. Using inducible cMYC overexpression, we demonstrate that post-pregnancy MECs are resistant to the downstream molecular programs induced by cMYC, a response that blunts carcinoma initiation, but does not perturb the normal pregnancy-induced epigenomic landscape. cMYC overexpression drives post-pregnancy MECs into a senescence-like state, and perturbations of this state increase malignant phenotypic changes. Taken together, our findings provide further insight into the cell-autonomous signals in post-pregnancy MECs that underpin the regulation of gene expression, cellular activation, and resistance to malignant development.

The developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.

Drug resistance to our current stock of antibiotics is projected to increase to levels that threaten our ability to reduce and eliminate bacterial infections, which is now considered one of the primary health care crises of the 21st century. Traditional antibiotic agents (e.g., penicillin) paved the way for massive advances in human health, but we need novel strategies to maintain the upper hand in the battle against pathogenic bacteria. Nontraditional strategies, such as targeted immunotherapies, could prove fruitful in complementing our antibiotic arsenal.

Pregnancy reprograms mammary epithelial cells (MECs) to control their responses to pregnancy hormone re-exposure and carcinoma progression. However, the influence of pregnancy on the mammary microenvironment is less clear. Here, we used single-cell RNA sequencing to profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis indicates an expansion of gd natural killer T-like immune cells (NKTs) following pregnancy and upregulation of immune signaling molecules in post-pregnancy MECs. We show that expansion of NKTs following pregnancy is due to elevated expression of the antigen-presenting molecule CD1d on MECs. Loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKTs, results in mammary oncogenesis. Collectively, our findings illustrate how pregnancy-induced changes modulate the communication between MECs and the immune microenvironment and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

17Pregnancy leaves a series of cellular and molecular modifications on mammary epithelial cells 18 (MECs). Pregnancy is also known for decreasing the predisposition of rodent and human MECs 19 to oncogenesis. Here, in order to understand the molecular basis for this effect, we analyzed 20 epigenetic changes in the enhancer landscape of murine post-pregnancy MECs, together with 21 their effect on gene regulation, tissue development and oncogenesis. Using in vivo and in vitro 22 analyses, we found that completion of a pregnancy cycle changed the dynamics of cellular 23 proliferation and gene expression in response to a second pregnancy. Our results also 24 demonstrated that post-pregnancy MECs are resistant to the initial molecular programs driven 25 by cMYC overexpression, a response that blocked MEC proliferation but did not perturb the 26 pregnancy-induced epigenomic landscape. Overall, our findings suggest that pregnancy-27 induced mammary cancer prevention involves the epigenomic changes in MECs brought about 28 by pregnancy. 29 30 89 This procedure induces mammary gland development that closely resembled both the 90 histological and epigenetic modifications observed in mice where pregnancy occurred following 91 conception [25] ( Supplementary Fig. 1a). 92Pre-and post-pregnancy MECs had similar transcription programs, as revealed by 93 unsupervised expression clustering, suggesting that their epithelial identity during tissue 94 4 homeostasis is not substantially altered by a previous pregnancy cycle. MECs harvested during 95 the early stages of a second pregnancy (D6) clustered together with those harvested at a later 96 time-point during a first pregnancy (D12), supporting that post-pregnancy MECs respond 97 robustly to consecutive pregnancy signals ( Fig.1a). In order to address whether this robust 98 response to second pregnancy signals have an epigenetic basis, we investigated the genomic 99 distribution of the active histone mark H3K27ac, which annotates active regulatory elements 100 (promoters and enhancers) in mammalian genomes, in the same cohort of MECs utilized for the 101 RNA-seq analysis ( Supplementary Fig. 1a). 102Post-pregnancy MECS exhibited an eight-fold increase in the number of exclusive H3K27ac 103 peaks (n=169,950), compared to pre-pregnancy MECs (n=20,741), demonstrating that 104 pregnancy significantly expands the regulatory landscape of MECs ( Fig. 1b). H3K27ac peaks in 105 pre-pregnancy MECs were enriched in pathways associated with kinase activity, RNA binding 106 and stem cells ( Supplementary Fig. 1b), while in post-pregnancy MECs, the H3K27ac peaks 107 enriched for pathways involved in regulation of cell polarity, mRNA splicing, DNA methylation 108 and response to stress ( Supplementary Fig. 1c). These observations show that pre- and post-109 pregnancy MECs engage in distinct cellular maintenance and tissue homeostasis pathways. 110Further investigation of the regulatory regions demarcated by the pregnancy-induced 111 epigenome demonstrated that the majority of H3K27ac pea...