An epigenetic memory of pregnancy in the mouse mammary gland

Abstract

Pregnancy is the major modulator of mammary gland activity. It induces a tremendous expansion of the mammary epithelium and the generation of alveolar structures for milk production. Anecdotal evidence from multiparous humans indicates that the mammary gland may react less strongly to the first pregnancy than it does to subsequent pregnancies. Here, we verify that the mouse mammary gland responds more robustly to a second pregnancy, indicating that the gland retains a long-term memory of pregnancy. A comparison of genome-wide profiles of DNA methylation in isolated mammary cell types reveals substantial and long-lasting alterations. Since these alterations are maintained in the absence of the signal that induced them, we term them epigenetic. The majority of alterations in DNA methylation affect sites occupied by the Stat5a transcription factor and mark specific genes that are upregulated during pregnancy. We postulate that the epigenetic memory of a first pregnancy primes the activation of gene expression networks that promote mammary gland function in subsequent reproductive cycles. More broadly, our data indicate that physiological experience can broadly alter epigenetic states, functionally modifying the capacity of the affected cells to respond to later stimulatory events.

Figure 1. Increased response of the mammary gland during a second pregnancy

(A) Experimental design. Nulliparous and parous mice were implanted with slow-release estrogen/progesterone pellets. Mammary glands from pellet-bearing mice were harvested at day 6 (D6) and day 12 (D12) post pellet implantation. (B) Whole mount images from pellet-bearing nulliparous and parous mice. Mammary glands were harvested, fixed and cleared prior to Carmine staining. (C) Glands from pellet-bearing nulliparous and parous mice were stained with an a-milk protein antibody.

Figure 2. Genome-wide methylation profiles of mammary epithelial cells

(A) Hierarchical clustering of genome-wide methylation profiles of mammary epithelial cells. Several other cell types are included for comparison (B) Relationship between compartment-specific HMRs and gene expression. The horizontal axes correspond to distances from transcription start sites (TSS) of genes with preferential expression in basal or luminal compartments. The height of each bar corresponds to the frequency of compartment-specific HMRs expressed genes.

Figure 3. Pregnancy leaves an epigenetic memory

(A) Hierarchical clustering of genome-wide methylation profiles from cells isolated from nulliparous and parous mice. (B) Representative example of parity-induced DNA methylation changes at the Birc2 gene locus. (C) Top 10 transcription factor motifs significantly enriched at luminal parous DMRs. (D) Bar graph showing the percentage of luminal HMRs, along with nulliparous- and parous-specific DMRs, that overlap with Stat5a peaks. (E) Occupancy heatmap showing the distribution of Stat5a peaks at parous DMRs; the rows correspond to Stat5a occupancy across the parous luminal DMRs (+/−3kb from DMR center); parous DMRs were sorted according to size (top=larger; bottom= shorter). The red lines correspond to Stat5a peaks and the gray lines represent the genomic regions spanned by DMRs.

Figure 4. An epigenetic memory primes genes for response in subsequent pregnancy

(A) The illustration shows the presence of nulliparous (green) and parous (red) DMRs within 4Kb of genes with role during lactation and involution. Promoter regions with both nulliparous and parous DMRs are represented in blue (present in both). Promoter regions with neither nulliparous nor parous DMRs are represented in white (no DMRs). (B) Average DNA methylation levels of parous luminal DMRs in nulliparous luminal cells, and luminal cells 2 and 12 months after pregnancy. (C) Pregnancy hormones provoke enhanced activation of genes associated with parous DMRs. qPCR analysis is shown for nulliparous and parous mice at day 6 and 12 after implantation of hormone pellets. All changes were significant to at least p<0.05.