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    • A complimentary approach with Per Luc

    2022-07-26

    A complimentary approach with Per2:Luc 17-DMAG sale exposed to hypoxia as well as with mBmal1:Luc circadian reporter cells in which we overexpressed variants of HIF-1α and HIF-2α revealed that hypoxia and HIFs can not only shorten the period but also decrease the amplitude of the clock (Figures 2 and S1). The amplitude effects of the HIFs seen here are in line with previous studies in which C2C12 and U2OS cells were treated with DMOG (Wu et al., 2017, Peek et al., 2017). Interestingly, our data appeared to be in contrast to one study (Wu et al., 2017) wherein overexpression of a degradation-resistant HIF1α (HIF1α-P402A/P564A) resulted in a lengthening of the period of up to 4 h in U2OS cells. The reasons for the discrepancies might be multiple and could be attributable to the different model systems used (U2OS osteosarcoma cells there versus non-cancer mammalian fibroblasts in our study) or the different way of cell synchronization (dexamethasone versus forskolin in our study). In addition, the other study used the doxycycline-dependent tetR system to overexpress HIF-1α in U2OS cells. Hence, secondary effects of the doxycycline treatment, which are known to inhibit protein translation in mitochondria and to impair metabolism may lead to effects that can confound experimental results (Moullan et al., 2015, Chatzispyrou et al., 2015). Nonetheless, and despite the differences, both studies underline the important roles of HIF-1α as circadian modulator. Although no study, including the present, can yet explain the detailed mechanism by which HIFs and DMOG affect the period, it is tempting to speculate that this may be mediated by the proposed effects of HIF on BMAL1 (Peek et al., 2017). Indeed, deletion of HIF-1α resulted in a dramatic reduction in BMAL1 mRNA levels (Figure S7) in line with another study wherein overexpression of HIF-1α induced it (Yu et al., 2015). However, HIFs and DMOG appear to act by a different mechanism when seeing the effects on the period. In the present and previous studies (Wu et al., 2017, Peek et al., 2017) DMOG lengthened the period (Figure S1), whereas overexpression of HIFs shortened it (Figures 2 and S1). The difference in period length between DMOG and HIFs is explainable by the rather pleiotropic effects of DMOG, which is a synthetic analog of α-ketoglutarate/2-oxoglutarate. As such, DMOG can act as a pan-inhibitor of α-ketoglutarate/2-oxoglutarate-dependent dioxygenases, a family consisting of about ∼70 enzymes in humans, among them the HIF-prolyl 4-hydroxylases (Hirsila et al., 2003, Kietzmann et al., 2017). Consequently, a number of HIF-independent effects may influence the data achieved upon usage of DMOG. In line are the results obtained upon knockdown of the α-ketoglutarate/2-oxoglutarate-dependent dioxygenases FIH-1 and the EglN family, which also resulted in a lengthened period (Wu et al., 2017). To circumvent the pleiotropic DMOG effects, we used a hydroxylation-resistant HIF-1α variant (PPN) to specify whether it can mimic the shorter period as seen during chronic hypoxia. Importantly, both HIF-1α and HIF-2α could resemble the effects of hypoxia on the period (Figures 2 and S1). Thus the difference in period length upon DMOG usage or EglN and FIH-1 knockdown versus hypoxia or HIF overexpression as seen in this study can be attributed to non-HIF targets of which several, so far unknown, may be involved in clock regulation (Ivan and Kaelin, 2017). Together, all these data suggest that the hypoxia-signaling pathway may have different layers that can affect the circadian period and amplitude. Intrigued by the HIF effects on the circadian period, and given that CRY proteins represent dominant critical regulators of the circadian period length in humans and mice (Van Der Horst et al., 1999, Vitaterna et al., 1999, Li et al., 2016, Hirota et al., 2012, Ode et al., 2017, Oshima et al., 2015, Siepka et al., 2007, Patke et al., 2017) we hypothesized a connection between HIFs and CRYs. Therefore, we probed whether CRY1 and CRY2 can interact with HIF-1α and HIF-2α, and indeed our experiments revealed that both CRY proteins were able to undergo interaction with the two HIF proteins (Figures 4 and S2).