Nuclear trafficking dynamics of Bromodomain-containing protein 7 (BRD7), a switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex subunit, in porcine oocytes and cleavage-stage embryos.

In the work presented here, we investigated how bromodomain-containing protein 7 (BRD7), a subunit associated with switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complexes, is trafficked between cellular compartments during embryo development. SWI/SNF complexes are multi-subunit complexes that contain a core catalytic subunit (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4, or member 2; SMARCA4 or SMARCA2) and a collection of additional subunits that guide the complexes to their appropriate loci; BRD7 is one of these additional subunits. We hypothesised that BRD7 is exported from the nuclei of porcine oocytes and embryos in a Chromosome Region Maintenance 1 (CRM1)-dependent manner and imported into the nuclei using the karyopherin α/ß1 heterodimer. Porcine oocytes and embryos were treated with inhibitors of CRM1-mediated nuclear export and karyopherin α/ß1-mediated nuclear import to test this hypothesis. An RNA interference assay and a dominant negative overexpression assay were also performed to determine if karyopherin α7 serves a specific role in BRD7 trafficking. Our findings indicate that BRD7 shuttles between nuclear and cytoplasmic compartments during cleavage development. The shuttling of BRD7 indicates that it serves a unique role in remodelling chromatin during this developmental window.

CLOCK regulates mammary epithelial cell growth and differentiation.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.