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.

Myeloid cell heterogeneity remains poorly studied in breast cancer, and particularly in premalignancy. Here, we used single cell RNA sequencing to characterize macrophage diversity in mouse pre-invasive lesions as compared to lesions undergoing localized invasion. Several subpopulations of macrophages with transcriptionally distinct profiles were identified, two of which resembled macrophages in the steady state. While all subpopulations expressed tumor-promoting genes, many of the populations expressed pro-inflammatory genes, differing from reports in tumor-associated macrophages. Gene profiles of the myeloid cells were similar between early and late stages of premalignancy, although expansion of some subpopulations occurred. These results unravel macrophage heterogeneity in early progression and may provide insight into early intervention strategies that target macrophages.

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.

Objectives Oxytocin (OXT) is widely used to facilitate labor. However, little is known about the effects of perinatal OXT exposure on the developing brain. We investigated the effects of maternal OXT administration on gene expression in perinatal mouse brains. Methods Pregnant C57BL/6 mice were treated with saline or OXT at term (n=6–7/group). Dams and pups were euthanized on gestational day (GD) 18.5 after delivery by C-section. Another set of dams was treated with saline or OXT (n=6–7/group) and allowed to deliver naturally; pups were euthanized on postnatal day 9 (PND9). Perinatal/neonatal brain gene expression was determined using Illumina BeadChip Arrays and real time quantitative PCR. Differential gene expression analyses were performed. In addition, the effect of OXT on neurite outgrowth was assessed using PC12 cells. Results Distinct and sex-specific gene expression patterns were identified in offspring brains following maternal OXT administration at term. The microarray data showed that female GD18.5 brains exhibited more differential changes in gene expression compared to male GD18.5 brains. Specifically, Cnot4 and Frmd4a were significantly reduced by OXT exposure in male and female GD18.5 brains, whereas Mtap1b, Srsf11, and Syn2 were significantly reduced only in female GD18.5 brains. No significant microarray differences were observed in PND9 brains. By quantitative PCR, OXT exposure reduced Oxtr expression in female and male brains on GD18.5 and PND9, respectively. PC12 cell differentiation assays revealed that OXT induced neurite outgrowth. Conclusions Prenatal OXT exposure induces sex-specific differential regulation of several nervous system-related genes and pathways with important neural functions in perinatal brains.

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...