In contrast, their blood triglyceride levels were 62% higher, consistent with Li et al.’s findings.24 Serum shock has been demonstrated

to induce rhythmic clock gene expression in cultured cells and thus provides a useful in vitro model to study clock mechanism. Based on the above observations, we extended our study Selleck Cisplatin and further investigated the function of BAF60a in clock machinery in cultured cells. We similarly used knockdown BAF60a expression in HepG2 cells and then exposed these cells to brief serum shock for 1 hour followed by observation over a period of 30 hours. Serum shock led to robust oscillation of Bmal1, Clock, Rev-erbα, Per1, and Per2 expression in control cells (Fig. 3). In contrast, rhythmic expression of these genes was essentially abolished in cells with RNA interference (RNAi) knockdown NVP-LDE225 of BAF60a. Of note, the expression of Bmal1, Per2, and BAF60a showed semidiurnal rather than diurnal rhythms in our results. One possible explanation is that the HepG2 we used here is a cancer cell line and in many cancers the circadian clock systems are disrupted and the

expression of the circadian clock genes does not exhibit regular rhythmicity any more.27 However, our purpose in this experiment was to assess the impact of BAF60a abolishment on the expression of circadian clock (although not the normal circadian clock) and our results indeed showed that BAF60a is essential for the maintenance of circadian gene expression. To identify transcription factors

that mediate the regulation of clock genes by BAF60a we examined the ability of BAF60a to synergize with these factors in the regulation of Bmal1 transcription. BAF60a dramatically augmented the transcriptional activity of RORα, but not RORγ, on a Bmal1 promoter reporter (Fig. 4A; Supporting Fig. 3). The synergistic effects of RORα and BAF60a were abolished when the ROR-binding sites (RORE) on the proximal Bmal1 promoter were mutated. This functional synergy between RORα and BAF60a was also observed for the endogenous Bmal1 gene (Fig. 4B). Furthermore, coimmunoprecipitation (CoIP) assays indicated that BAF60a and RORα had physical interaction and formed a complex in vivo (Fig. 4C). Their interaction was confirmed in the liver at ZT1 when BAF60a expression is high 上海皓元医药股份有限公司 (Fig. 4D). Previous studies indicated that Rev-erbα negatively regulates Bmal1 transcription by recruiting corepressor proteins. Consistent with this, both Rev-erbα and Rev-erbβ drastically repressed the stimulatory effects of BAF60a on the Bmal1-luc reporter (Fig. 4E). These results illustrate that the ability of BAF60a to activate Bmal1 transcription is modulated by the relative abundance of the RORα and Rev-erb family of orphan receptors. ChIP assays in HepG2 cells indicated that BAF60a was present near RORE on the proximal Bmal1 promoter (Fig. 4F; Supporting Fig. 5).