Several recent studies have linked clock function with the

Several recent studies have linked clock function with the cell cycle and reported that clock components, such as PER1, PER2, BMAL1, and CRY1/2 decrease cell proliferation or improve the action of anti-cancer drugs in different cancer cell lines. Moreover, certain types of human cancer show an altered expression of circadian clock genes (reviewed in Shostak, 2017). In addition to disturbances of the circadian clock, most, if not all, solid tumors display hypoxic areas. Hypoxia has been shown to be the major driver of tumor angiogenesis and a critical determinant for proliferation, cell growth, and apoptosis (Semenza, 2017, Masson and Ratcliffe, 2014, Kaelin and Ratcliffe, 2008). Mechanistically, a group of the bHLH-PAS family transcription factors called hypoxia-inducible transcription factors, among which hypoxia-inducible factor (HIF)-1α is the best characterized, mediate the transcriptional SecinH3 to hypoxia. Thereby, HIF-1α together with its binding partner HIF-1β (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) binds to hypoxia-response elements (HREs) within the promoters of numerous hypoxia-responsive genes (Semenza, 2017, Masson and Ratcliffe, 2014, Kaelin and Ratcliffe, 2008). The disruption of the circadian rhythm in patients with cancer and the appearance of hypoxia in tumors raises the question whether and how the deregulation of the circadian clock system has an impact on the hypoxia response. Several findings suggested a cross talk between the circadian clock and the hypoxia signaling pathway (Hogenesch et al., 1998, Chilov et al., 2001, Eckle et al., 2012), but the mechanisms behind are not completely understood and have just started to emerge. Recent reports have shown that the modulation of oxygen levels can reset the circadian clock at the positive limb in a HIF-1α-dependent manner (Adamovich et al., 2017) and that HIF-1α and BMAL1 engage in a synergistic cross talk (Wu et al., 2017), which adapts anaerobic metabolism in skeletal muscle (Peek et al., 2017). Although these findings favor the view that this cross talk is bidirectional and would eventually also involve the negative arm of the circadian key players, their participation, in particular that of CRY proteins, remains unknown. In the current study we broaden this view by showing that CRY1, but not CRY2, acts as a repressor of HIFs. This occurs via a specific protein-protein interaction that reduces the binding of HIFs at the HREs of target gene promoters and by altering HIF half-life. Disruption of the CRY1-HIF cross talk at the cellular level shows that CRY1 and HIF-1α have an opposite action on cell growth.
Results
Discussion The current study extends recently reported findings on the interplay between the circadian clock and the hypoxia pathway (Adamovich et al., 2017, Wu et al., 2017, Peek et al., 2017) by providing detailed mechanistic insights indicating how the circadian clock component CRY1 can regulate the hypoxia response pathway at the level of HIF-1α and HIF-2α. In particular we show that (1) CRY1 directly interacts with both HIF-1α and HIF-2α, (2) that the interaction of CRY1 with HIFs masks the DNA-binding domain of HIFs and consequently changes HIF DNA binding and expression of HIF target genes, and (3) that the CRY1-HIFα interaction is important for cellular growth. By broadening the knowledge about CRY1's involvement in more than the circadian clock pathway, the current study also extends the existing information about the interplay between the circadian clock and the hypoxia response pathway. Although this cross talk was proposed on the basis of in vitro findings almost 20 years ago (Hogenesch et al., 1998), a functional validation presenting a reciprocal regulation between the positive clock limb protein BMAL1 and HIF-1α (Peek et al., 2017) was just recently reported while this study on the negative limb was ongoing. Our data show that hypoxia can modulate the circadian rhythm in vivo and in vitro. This is in line with findings of a recent study demonstrating that mimicry of circadian physiological oxygen rhythms as occurring in mouse blood can affect the setting of the clock (Adamovich et al., 2017). In addition, we show that mice under chronic hypoxia become arrhythmic in total darkness (DD) indicating more severe effects of hypoxia on the clock in general (Figure 1). Although the reasons are not known yet, these activity data may be the result of a partial uncoupling of central and peripheral clocks due to disturbed adaptation to chronic hypoxia similar to that seen with restricted feeding (Damiola et al., 2000). This may express as arrhythmicity in periods coinciding with an increased oxygen consumption (Adamovich et al., 2017) such as the onset of activity and food ingestion, which in mice normally occur during the dark phase of the daily rhythm.