Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Maternal high-fat diet exposure during gestation, lactation, or gestation and lactation differentially affects intestinal morphology and proteome of neonatal mice.

Offspring nutrition depends on the mother during gestation and lactation; thus, maternal nutrition and metabolism can affect their development. We hypothesized that maternal exposure to high-fat (HF) diet affects neonate's gastrointestinal tract development. Our objective was to determine the effect of maternal HF diet during gestation and lactation on neonate's duodenum histomorphology and proteome. Female mice were fed either a control (C, 10% kcal fat) or an HF (60% kcal fat) diet for 4 weeks and bred. On postnatal day 2, half the pups were cross-fostered to dams fed on different diet, creating 4 treatments: C-C, C-HF, HF-C, and HF-HF, indicating maternal diet during gestation-lactation, respectively. On postnatal day 12, pups' duodenum was excised and prepared for histology and liquid chromatography-tandem mass spectrometry analysis of proteome. Villi were significantly longer in HF-HF pups, and crypt cell proliferation rate was not different among treatments. Between C-C and HF-HF, HF-C, or C-HF, 812, 601, or 894 proteins were differentially expressed (Tukey adjusted P < .05), respectively. Functional analysis clustered proteins upregulated in HF-HF vs C-C in fat digestion and absorption, extracellular matrix, cell adhesion, immune response, oxidation-reduction processes, phagocytosis, and transport categories. Proteins downregulated were classified as RNA splicing, translation, protein folding, endocytosis, and transport. There was evidence for a carryover effect of exposure to HF diet during gestation to the postnatal period. Alterations in proteome relative to HF exposure potentially reflect long-term changes in the functioning of the duodenum.