The CRY protein contains a conserved
The CRY1 protein contains a conserved photolyase homology region crucially involved in repression of CLOCK/BMAL1; a C-terminal helix also known as the predicted coiled coil (CC), which interacts with PER2 and FBXL3 in a mutually exclusive manner; and a C-terminal extension also referred to as the “tail” (Figure 5) (Chaves et al., 2011, Merbitz-Zahradnik and Wolf, 2015). Our mapping experiments showed that HIF-1α interacts with the CRY1 “tail” region, a part of the protein (Figure 5), which has the ability to modulate the period length and amplitude of the resulting oscillation (Khan et al., 2012, Xu et al., 2015). In line, a recent report showed that a dominant CRY1 allele coding for a protein with a deletion of 24 residues in the tail region (Patke et al., 2017) caused lengthening of the circadian period and could be linked to familial delayed sleep phase disorder. Thus the effects seen under hypoxia on the period length are in accord with the interaction of HIF-1α with CRY1's tail region. Furthermore, the tail has been shown to affect CRY1 translocation, to interact with the BMAL1 transactivation domain possibly in an acetylation-dependent fashion, and to be phosphorylated in a manner that involves regulation by DNA-PK (Xu et al., 2015, Chaves et al., 2006, Czarna et al., 2011, Hirayama et al., 2007). In addition, the existence of a nuclear localization signal in the CRY1 tail coincides well with the nuclear localization of its interaction with HIFs as seen in the BiFC assays (Figures 4 and S2). In line, the bHLH domain of HIF-1α, which appears to work as the CRY1 interaction module (Figure 6), also contains a nuclear localization sequence (Kallio et al., 1998). Although not directly tested in this study, a similar mode of interaction is likely occurring with HIF-2α because both share an amino pterostilbene sequence identity of ∼85% in the bHLH domain. As the HIF bHLH domain is primarily involved in DNA binding, it is conceivable that the CRY1 tail could affect binding of HIFs to target gene promoters causing a repression of their expression. Thereby the repressive effect of CRY1 may not only depend solely on HIF-1α but also on the promoter context. This is exemplified with the PAI-1 promoter where CRY1 did not repress its activity under normoxia, whereas it clearly suppressed its hypoxia-dependent induction. To minimize the influence of the promoter context and to generalize the findings with respect to HIFs, we then used a bona fide hypoxia reporter containing three HREs as enhancers in front of the SV40 promoter and the luciferase gene. Indeed, these HRE reporter gene assays revealed that CRY1 suppressed their activity under normoxia and their hypoxia and HIF-1-dependent induction. Vice versa, the ChIP experiments from CRY1-deficient cells showed enhanced binding of HIFs to target gene promoters under both normoxia and hypoxia (Figures 7D and S5). Together, these effects need to be seen in context with our data showing that absence of CRY1 regulates HIF-1α at two levels, namely, by increasing its transcription and its stability (Figure 8). Consequently, the net effect is an increase in HIF-1α binding to its enhancers under both conditions. Overall, these data point to a suppressive role of CRY1, and in particular its tail, in the transcriptional response toward hypoxia at various stages of the circadian cycle. The current data are also in agreement with another recent study also showing enhanced HIF-1α levels in CRY-deficient cells, whereas BMAL1 absence decreased HIF-1α levels (Peek et al., 2017). Similarly, Per2 supported recruitment of HIF-1α to the VEGF promoter (Kobayashi et al., 2017). Together with reports from mouse liver and cardiac tissue, showing that Hif1α mRNA levels cycled in a circadian manner (Eckle et al., 2012, Wu et al., 2017) and data showing a rhythmic appearance of nuclear HIF1α in mouse brain and kidney (Adamovich et al., 2017), the current findings substantiate the existence of a feedback mechanism in which the circadian clock pathway can regulate HIF-1α expression.