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

Abstract

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.

Circadian clocks and their integration with metabolic and reproductive systems: our current understanding and its application to the management of dairy cows

“…Mammary core clock genes’ expression was also shifted between day and night restricted feeding which was accompanied by changes in the timing of de novo fatty acid synthesis in the gland ( Salfer and Harvatine, 2020 ). In support of a role of mammary clock in regulating fatty acid synthesis and metabolism, our ChIP-seq analysis of transcriptional targets of BMAL1 in mouse mammary epithelial cells identified genes that regulate uptake, transport and synthesis of lipids ( Casey et al, 2021 ). Moreover, genes that regulate fatty acid synthesis were shown to exhibit circadian rhythms of expression in lactating mammary tissue ( Maningat et al, 2009 ), and our studies of mid-lactation cows found circadian disruption due to exposure to continuous light-dark phase shifts decreased expression of fatty acid synthase (FASN ) and acetyl CoA-carboxylase (ACACA) in the mammary gland ( Casey et al, 2014a ).…”

“…Ruminants rely on gluconeogenesis for 90% of their glucose supply ( Young, 1977 ), and thus minimizing exposures to factors that disrupt circadian clocks may be particularly important in late gestation and early lactation cows, when glucose requirements are particularly high ( Bell and Bauman, 1997 ). Limiting exposure to chronodisruptors may also decrease the risk for developing diseases in the peripartum period, as global analysis of the impact of exposing late gestation dairy cows to circadian disruption on hepatic transcriptome and muscle proteome found a potential for an increased risk of developing fatty liver ( Casey et al 2021 ), and increased oxidative stress in muscle tissue ( McCabe et al, 2021a ).…”

“…Our studies of women found lower sleep efficiency and night-to-night variation in sleep, which disrupt circadian clocks, was related to a higher risk for delayed lactogenesis II (secretory activation) ( Casey et al, 2019 ). ChIP-seq analysis of transcriptional targets of BMAL1 support a role for the mammary epithelial clock in regulation of genes that regulate milk synthesis, growth and differentiation of epithelial cells as well as the coordination of hormonal signals with nutrient uptake, which is needed to initiate lactation ( Casey et al, 2021 ). Moreover, in 2D cultures of BMAL1 knock-out cells there was an increased rate of cell death related increased levels of reactive oxygen species and lower expression of super-oxide dismutase 3 (SOD3).…”

BMAL1 and CLOCK control polarization and lumen formation during mammary alveologenesis in 3D culture

“…The normal mouse mammary epithelial cell line, HC11, is frequently used for functional studies to understand the role of genes, pathways, and systems related to the regulation of growth, differentiation, and acini morphogenesis. [12][13][14] To understand the role of BMAL1 and CLOCK in the regulation of mammary epithelial cell growth and differentiation, HC11 lines with BMAL1 knock out (BMAL1-KO) 5 using CRISPR-Cas9 and CLOCK protein reduction using shRNA technology (shCLOCK) 10 were established and deletion and knockdown were confirmed in previous experiments. The differing systems to knockout (remove) or knockdown of proteins enables the study and identification of non-circadian clock functions of the target gene, as well as reduced function of the core circadian clock in the cells.…”

“…Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of BMAL1 target genes in the mouse mammary epithelial cell line, HC11, found that among the potential BMAL1 targets were genes that regulate mammary morphogenesis. 5 These include genes that regulate cell division, cell-cell adhesion molecules, and cell-extracellular matrix (ECM) adhesion molecules. Moreover, multiple BMAL1 target genes function to effect cell polarity including molecules that regulate ion and substrate transport across cells.…”