FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2919465720> ?p ?o ?g. }

• W2919465720 endingPage "304" @default.
• W2919465720 startingPage "284" @default.
• W2919465720 abstract "•Hypoxia and HIFs affect the circadian rhythm•CRY1 directly interacts with both HIF-1α and HIF-2α•CRY1 inhibits binding of HIFs to its target gene promoters•The CRY1-HIFα interaction has opposite roles on cellular growth and migration The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis. The circadian clock and the hypoxia-signaling pathway are regulated by an integrated interplay of positive and negative feedback limbs that incorporate energy homeostasis and carcinogenesis. We show that the negative circadian regulator CRY1 is also a negative regulator of hypoxia-inducible factor (HIF). Mechanistically, CRY1 interacts with the basic-helix-loop-helix domain of HIF-1α via its tail region. Subsequently, CRY1 reduces HIF-1α half-life and binding of HIFs to target gene promoters. This appeared to be CRY1 specific because genetic disruption of CRY1, but not CRY2, affected the hypoxia response. Furthermore, CRY1 deficiency could induce cellular HIF levels, proliferation, and migration, which could be reversed by CRISPR/Cas9- or short hairpin RNA-mediated HIF knockout. Altogether, our study provides a mechanistic explanation for genetic association studies linking a disruption of the circadian clock with hypoxia-associated processes such as carcinogenesis. A number of epidemiological studies showed that disturbances of the circadian rhythm strongly correlate with carcinogenesis and tumor progression in humans (for review see Fu and Lee, 2003Fu L. Lee C.C. The circadian clock: pacemaker and tumour suppressor.Nat. Rev. Cancer. 2003; 3: 350-361Crossref PubMed Scopus (589) Google Scholar, Filipski and Levi, 2009Filipski E. Levi F. Circadian disruption in experimental cancer processes.Integr. Cancer Ther. 2009; 8: 298-302Crossref PubMed Scopus (97) Google Scholar, Sahar and Sassone-Corsi, 2009Sahar S. Sassone-Corsi P. Metabolism and cancer: the circadian clock connection.Nat. Rev. Cancer. 2009; 9: 886-896Crossref PubMed Scopus (410) Google Scholar, Rana and Mahmood, 2010Rana S. Mahmood S. Circadian rhythm and its role in malignancy.J. Circadian Rhythms. 2010; 8: 3Crossref PubMed Scopus (87) Google Scholar). Accordingly, the International Agency for Research on Cancer classified shift work as a group 2A carcinogenic factor (Straif et al., 2007Straif K. Baan R. Grosse Y. Secretan B. Ghissassi F.E. Bouvard V. Altieri A. Benbrahim-Tallaa L. Cogliano V. WHO International Agency for Research on Cancer Monograph Working GroupCarcinogenicity of shift-work, painting, and fire-fighting.Lancet Oncol. 2007; 8: 1065-1066Abstract Full Text Full Text PDF PubMed Google Scholar). The circadian rhythm in mammals is maintained by an integrated network of the central (neural or brain) and peripheral (tissue-specific) clocks. The central clock is located in the suprachiasmatic nucleus of the brain, receives light cues to keep in phase with the light-dark cycle, and synchronizes the peripheral clocks in various tissues through a variety of electrical, endocrine, and metabolic signaling pathways (Albrecht, 2012Albrecht U. Timing to perfection: the biology of central and peripheral circadian clocks.Neuron. 2012; 74: 246-260Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, Richards and Gumz, 2012Richards J. Gumz M.L. Advances in understanding the peripheral circadian clocks.FASEB J. 2012; 26: 3602-3613Crossref PubMed Scopus (126) Google Scholar). At the genetic level, both the central and the peripheral clocks are regulated by an interplay of positive and negative feedback loops involving the same set of clock genes (Yagita et al., 2001Yagita K. Tamanini F. Van der Horst G.T.J. Okamura H. Molecular mechanisms of the biological clock in cultured fibroblasts.Science. 2001; 292: 278-281Crossref PubMed Scopus (373) Google Scholar). Thereby, the bHLH-PAS transcription factors CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain-muscle Arnt-like protein 1) represent the major components of the core clock's positive limb. They induce, among others, the expression of the proteins PER (period 1,2) and CRY (cryptochrome 1,2), which constitute the major arm of the negative limb. The induced PER and CRY proteins then form a complex with each other as well as with modulator proteins such as CK1ε, CKII, or FBXL3 and act as repressors of CLOCK/BMAL heterodimers. Subsequently, they inhibit their own expression as well as those of other CLOCK/BMAL-regulated output genes (Griffin et al., 1999Griffin Jr., E.A. Staknis D. Weitz C.J. Light-independent role of CRY1 and CRY2 in the mammalian circadian clock.Science. 1999; 286: 768-771Crossref PubMed Scopus (515) Google Scholar, Kume et al., 1999Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar, Yoo et al., 2005Yoo S.H. Ko C.H. Lowrey P.L. Buhr E.D. Song E.J. Chang S. Yoo O.J. Yamazaki S. Lee C. Takahashi J.S. A noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo.Proc. Natl. Acad. Sci. U S A. 2005; 102: 2608-2613Crossref PubMed Scopus (238) Google Scholar, Sato et al., 2006Sato T.K. Yamada R.G. Ukai H. Baggs J.E. Miraglia L.J. Kobayashi T.J. Welsh D.K. Kay S.A. Ueda H.R. Hogenesch J.B. Feedback repression is required for mammalian circadian clock function.Nat. Genet. 2006; 38: 312-319Crossref PubMed Scopus (290) Google Scholar). The core loop is interconnected to several other feedback loops, including those of REV-ERBs or PPARα/RORs, which repress or activate BMAL1 expression, respectively (Albrecht, 2012Albrecht U. Timing to perfection: the biology of central and peripheral circadian clocks.Neuron. 2012; 74: 246-260Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, Ko and Takahashi, 2006Ko C.H. Takahashi J.S. Molecular components of the mammalian circadian clock.Hum. Mol. Genet. 2006; 15: R271-R277Crossref PubMed Scopus (1220) Google Scholar, Asher and Schibler, 2011Asher G. Schibler U. Crosstalk between components of circadian and metabolic cycles in mammals.Cell Metab. 2011; 13: 125-137Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Overall, the continuous interplay between the core CLOCK/BMAL1 positive limb and the PER/CRY negative limb in concert with post-translational modifications and interconnected loops results in the oscillation of gene expression in a circadian manner. 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, 2017Shostak A. Circadian clock, cell division, and cancer: from molecules to organism.Int. J. Mol. Sci. 2017; 18https://doi.org/10.3390/ijms18040873Crossref PubMed Scopus (102) Google Scholar). 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, 2017Semenza G.L. Hypoxia-inducible factors: coupling glucose metabolism and redox regulation with induction of the breast cancer stem cell phenotype.EMBO J. 2017; 36: 252-259Crossref PubMed Scopus (221) Google Scholar, Masson and Ratcliffe, 2014Masson N. Ratcliffe P.J. Hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways.Cancer Metab. 2014; 2: 3Crossref PubMed Google Scholar, Kaelin and Ratcliffe, 2008Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.Mol. Cell. 2008; 30: 393-402Abstract Full Text Full Text PDF PubMed Scopus (2192) Google Scholar). 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 adaptation 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, 2017Semenza G.L. Hypoxia-inducible factors: coupling glucose metabolism and redox regulation with induction of the breast cancer stem cell phenotype.EMBO J. 2017; 36: 252-259Crossref PubMed Scopus (221) Google Scholar, Masson and Ratcliffe, 2014Masson N. Ratcliffe P.J. Hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways.Cancer Metab. 2014; 2: 3Crossref PubMed Google Scholar, Kaelin and Ratcliffe, 2008Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.Mol. Cell. 2008; 30: 393-402Abstract Full Text Full Text PDF PubMed Scopus (2192) Google Scholar). 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., 1998Hogenesch J.B. Gu Y.Z. Jain S. Bradfield C.A. The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors.Proc. Natl. Acad. Sci. U S A. 1998; 95: 5474-5479Crossref PubMed Scopus (625) Google Scholar, Chilov et al., 2001Chilov D. Hofer T. Bauer C. Wenger R.H. Gassmann M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain.FASEB J. 2001; 15: 2613-2622Crossref PubMed Scopus (106) Google Scholar, Eckle et al., 2012Eckle T. Hartmann K. Bonney S. Reithel S. Mittelbronn M. Walker L.A. Lowes B.D. Han J. Borchers C.H. Buttrick P.M. et al.Adora2b-elicited Per2 stabilization promotes a HIF-dependent metabolic switch crucial for myocardial adaptation to ischemia.Nat. Med. 2012; 18: 774-782Crossref PubMed Scopus (229) Google Scholar), 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., 2017Adamovich Y. Ladeuix B. Golik M. Koeners M.P. Asher G. Rhythmic oxygen levels reset circadian clocks through HIF1a.Cell Metab. 2017; 25: 93-101Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and that HIF-1α and BMAL1 engage in a synergistic cross talk (Wu et al., 2017Wu Y. Tang D. Liu N. Xiong W. Huang H. Li Y. Ma Z. Zhao H. Chen P. Qi X. Zhang E.E. Reciprocal regulation between the circadian clock and hypoxia signaling at the genome level in mammals.Cell Metab. 2017; 25: 73-85Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), which adapts anaerobic metabolism in skeletal muscle (Peek et al., 2017Peek C.B. Levine D.C. Cedernaes J. Taguchi A. Kobayashi Y. Tsai S.J. Bonar N.A. McNulty M.R. Ramsey K.M. Bass J. Circadian clock interaction with HIF1a mediates oxygenic metabolism and anaerobic glycolysis in skeletal muscle.Cell Metab. 2017; 25: 86-92Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). 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. Overall, our study identifies a new level of control that links defects in the circadian clock system with hypoxia signaling and carcinogenesis. To study the possible interplay between the circadian clock and hypoxia we measured the circadian behavior of mice under normoxia (21% O2) and hypoxia (17% O2) at 12 h + 12 h light/dark (LD) cycles and at constant darkness (dark/dark; DD). When mice were kept under both normoxia and hypoxia at LD they showed circadian periodicity (Figures 1A and 1B). Intriguingly, we observed that mice at DD lost their circadian rhythm (Figures 1A and 1B) and expressed HIF target genes when exposed to hypoxia (Figure 1C). Together, these data indicate that hypoxia is able to modulate the circadian rhythm. As the hypoxia-mediated loss of the circadian rhythm in mice at DD stems from systemic and cell-autonomous oscillator effects, we next tested to what extent hypoxia affects the cell-autonomous clock. To do this, we first verified the effect of chronic hypoxia (3% O2 for 32 h) in synchronized Per2:Luc-transfected fibroblasts, which possess a cell-autonomous and self-sustained circadian clock (Yagita et al., 2001Yagita K. Tamanini F. Van der Horst G.T.J. Okamura H. Molecular mechanisms of the biological clock in cultured fibroblasts.Science. 2001; 292: 278-281Crossref PubMed Scopus (373) Google Scholar, Balsalobre et al., 1998Balsalobre A. Damiola F. Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells.Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1557) Google Scholar). We found that hypoxia affected the circadian rhythm in two ways, first by reducing the amplitude and second by shortening the period by about 30 min (Figures 2A and 2B). Given that the response to hypoxia is mainly mediated by an increased abundance of the transcription factors HIF-1α and HIF-2α, we next investigated how HIF-1α or HIF-2α affect the circadian clock. To do this, we first cotransfected an expression vector for a hypoxia-mimicking hydroxylation-resistant HIF-1α variant (P/P/N) (Flügel et al., 2012Flügel D. Görlach A. Kietzmann T. GSK-3beta regulates cell growth, migration, and angiogenesis via Fbw7 and USP28-dependent degradation of HIF-1alpha.Blood. 2012; 119: 1292-1301Crossref PubMed Scopus (136) Google Scholar) with the circadian rhythm reporter Bmal1:Luc and analyzed circadian rhythmicity. Similar to hypoxia, overexpression of HIF-1α shortened the circadian period and reduced the amplitude (Figures 2C and 2D). Overexpression of HIF-2α showed a similar response (Figures S1A and S1B). In contrast to hypoxia and HIFs, the non-specific prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG), which is often considered as a hypoxia mimetic, lengthened the period (Figures S1C and S1D). Together, these data indicate that HIF-1α and HIF-2α mimic the effect of hypoxia on the circadian clock, whereas the DMOG effects appear to be HIF independent. The aforementioned observations appear to be important for genes that are responsive to both hypoxia and the circadian clock. The SERPINE1 gene encoding plasminogen activator inhibitor-1 (PAI-1) fulfills these criteria and is well known to be a bona fide hypoxia-responsive and circadian-rhythm-regulated gene (Dimova et al., 2005Dimova E.Y. Moller U. Herzig S. Fink T. Zachar V. Ebbesen P. Kietzmann T. Transcriptional regulation of plasminogen activator inhibitor-1 expression by insulin-like growth factor-1 via MAP kinases and hypoxia-inducible factor-1 in HepG2 cells.Thromb. Haemost. 2005; 93: 1176-1184Crossref PubMed Scopus (37) Google Scholar, Kietzmann et al., 1999Kietzmann T. Roth U. Jungermann K. Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes.Blood. 1999; 94: 4177-4185Crossref PubMed Google Scholar, Maemura et al., 2000Maemura K. de la Monte S.M. Chin M.T. Layne M.D. Hsieh C.M. Yet S.F. Perrella M.A. Lee M.E. CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression.J. Biol. Chem. 2000; 275: 36847-36851Crossref PubMed Scopus (175) Google Scholar, Schoenhard et al., 2003Schoenhard J.A. Smith L.H. Painter C.A. Eren M. Johnson C.H. Vaughan D.E. Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2.J. Mol. Cell. Cardiol. 2003; 35: 473-481Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Wang et al., 2006Wang J. Yin L. Lazar M.A. The orphan nuclear receptor Rev-erb alpha regulates circadian expression of plasminogen activator inhibitor type 1.J. Biol. Chem. 2006; 281: 33842-33848Crossref PubMed Scopus (105) Google Scholar, Oishi et al., 2009Oishi K. Miyazaki K. Uchida D. Ohkura N. Wakabayashi M. Doi R. Matsuda J. Ishida N. PERIOD2 is a circadian negative regulator of PAI-1 gene expression in mice.J. Mol. Cell. Cardiol. 2009; 46: 545-552Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Cheng et al., 2011Cheng S. Jiang Z. Zou Y. Chen C. Wang Y. Liu Y. Xiao J. Guo H. Wang Z. Downregulation of Clock in circulatory system leads to an enhancement of fibrinolysis in mice.Exp. Biol. Med. (Maywood). 2011; 236: 1078-1084Crossref PubMed Scopus (8) Google Scholar). Therefore we investigated whether the hypoxia-induced shifts in circadian rhythmicity are transferred to the expression of hypoxia-responsive target genes and to what extent one or the other response prevails. To examine this, we analyzed PAI-1 expression on protein and reporter gene level in synchronized cells. We found that hypoxia enhanced PAI-1 expression and shortened the circadian period of PAI-1 by about 40 min (Figures 3A–3D). Together, these data further substantiate that hypoxia and HIFs can advance the period of clock-dependent genes, whereas the amplitude is likely also modulated depending on the transcriptional context. Accumulating genetic data indicate that the negative limb of the circadian regulators, in particular CRY1, is responsible for maintaining the period length or amplitude of the circadian clock and that CRY1 is the most potent repressor in the clock's negative limb (Griffin et al., 1999Griffin Jr., E.A. Staknis D. Weitz C.J. Light-independent role of CRY1 and CRY2 in the mammalian circadian clock.Science. 1999; 286: 768-771Crossref PubMed Scopus (515) Google Scholar, Kume et al., 1999Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar, Van Der Horst et al., 1999Van Der Horst G.T.J. Muijtjens M. Kobayashi K. Takano R. Kanno S. Takao M. De Wit J. Verkerk A. Eker A.P.M. Van Leenen D. et al.Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms.Nature. 1999; 398: 627-630Crossref PubMed Scopus (1120) Google Scholar, Vitaterna et al., 1999Vitaterna M.H. Selby C.P. Todo T. Niwa H. Thompson C. Fruechte E.M. Hitomi K. Thresher R.J. Ishikawa T. Miyazaki J. et al.Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2.Proc. Natl. Acad. Sci. U S A. 1999; 96: 12114-12119Crossref PubMed Scopus (563) Google Scholar, Li et al., 2016Li Y. Xiong W. Zhang E.E. The ratio of intracellular CRY proteins determines the clock period length.Biochem. Biophys. Res. Commun. 2016; 472: 531-538Crossref PubMed Scopus (19) Google Scholar, Liu et al., 2007Liu A.C. Welsh D.K. Ko C.H. Tran H.G. Zhang E.E. Priest A.A. Buhr E.D. Singer O. Meeker K. Verma I.M. et al.Intercellular coupling confers robustness against mutations in the SCN circadian clock network.Cell. 2007; 129: 605-616Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, Khan et al., 2012Khan S.K. Xu H. Ukai-Tadenuma M. Burton B. Wang Y. Ueda H.R. Liu A.C. Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function.J. Biol. Chem. 2012; 287: 25917-25926Crossref PubMed Scopus (58) Google Scholar, Matsuo et al., 2003Matsuo T. Yamaguchi S. Mitsui S. Emi A. Shimoda F. Okamura H. Control mechanism of the circadian clock for timing of cell division in vivo.Science. 2003; 302: 255-259Crossref PubMed Scopus (905) Google Scholar, Koike et al., 2012Koike N. Yoo S.H. Huang H.C. Kumar V. Lee C. Kim T.K. Takahashi J.S. Transcriptional architecture and chromatin landscape of the core circadian clock in mammals.Science. 2012; 338: 349-354Crossref PubMed Scopus (943) Google Scholar, Langmesser et al., 2008Langmesser S. Tallone T. Bordon A. Rusconi S. Albrecht U. Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK.BMC Mol. Biol. 2008; 9: 41Crossref PubMed Scopus (85) Google Scholar, Ye et al., 2014Ye R. Selby C.P. Chiou Y.Y. Ozkan-Dagliyan I. Gaddameedhi S. Sancar A. Dual modes of CLOCK: BMAL1 inhibition mediated by Cryptochrome and period proteins in the mammalian circadian clock.Genes Dev. 2014; 28: 1989-1998Crossref PubMed Scopus (128) Google Scholar, Michael et al., 2017Michael A.K. Fribourgh J.L. Chelliah Y. Sandate C.R. Hura G.L. Schneidman-Duhovny D. Tripathi S.M. Takahashi J.S. Partch C.L. Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1.Proc. Natl. Acad. Sci. U S A. 2017; 114: 1560-1565Crossref PubMed Scopus (56) Google Scholar). Based on CRY1's potency and responsibility for period and amplitude maintenance and the recent reports on the HIF-1α-BMAL1 cross talk (Wu et al., 2017Wu Y. Tang D. Liu N. Xiong W. Huang H. Li Y. Ma Z. Zhao H. Chen P. Qi X. Zhang E.E. Reciprocal regulation between the circadian clock and hypoxia signaling at the genome level in mammals.Cell Metab. 2017; 25: 73-85Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Peek et al., 2017Peek C.B. Levine D.C. Cedernaes J. Taguchi A. Kobayashi Y. Tsai S.J. Bonar N.A. McNulty M.R. Ramsey K.M. Bass J. Circadian clock interaction with HIF1a mediates oxygenic metabolism and anaerobic glycolysis in skeletal muscle.Cell Metab. 2017; 25: 86-92Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) as well as the aforementioned findings that HIFs shortened the period and affected the amplitude of the circadian clock, we hypothesized that HIFs would interact with CRY1. To examine this, we performed coimmunoprecipitation analyses with hypoxic mouse embryonic fibroblasts (MEFs). The assays revealed that CRY1 could be detected when HIF-1α was precipitated from the cells (Figure 4A ). Furthermore, CRY1 could also be detected upon precipitation with HIF-2α antibodies, although the binding signal was low because of the low abundance of HIF-2α (Figure S2A). Next, we verified these interactions and performed western blot analyses from immunoprecipitates of HEK293 cells expressing Myc-tagged HIF-1α, V5-tagged HIF-2α, and hemagglutinin (HA)-tagged CRY1. The blots from anti-Myc-tagged HIF-1α or anti-V5-tagged HIF-2α immunoprecipitations showed the presence of HA-tagged CRY1 when probed with the HA-tag antibody indicating interaction of HIFα proteins and CRY1 (Figures 4B and S2B). In addition, HIFα proteins were also able to interact with CRY2 (Figures 4C and S2C). We also employed a bimolecular fluorescence complementation (BiFC) assay to verify whether these interactions are direct. The BiFC assay takes advantage from the possibility that a fluorescent protein complex can be formed through association of two per se non-interacting and non-fluorescent N-terminal (YN) and C-terminal (YC) fragments of the yellow fluorescent protein (YFP). The complementation of the fluorescent complex is achieved when YN and YC are brought into proximity owing to a direct interaction of the proteins fused to the YN and YC fragments (Figure 4D) (Kerppola, 2008Kerppola T.K. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.Annu. Rev. Biophys. 2008; 37: 465-487Crossref PubMed Scopus (485) Google Scholar). We generated vectors allowing expression of fusion proteins consisting of HIF-1α or HIF-2α and the C-terminal part of the YFP (HIF-1α-YC or HIF-2α -YC) as well as of CRY1 and the N-terminal part of the YFP (CRY1-YN). In addition, constructs for the HIFα partner ARNT as well as for the CRY1 partner PER1 served as positive controls to indicate HIF-1α -ARNT or CRY1-PER1 interaction, respectively. The BiFC analyses revealed a strong fluorescent signal when either HIF-1α-YC (Figures 4E and 4F) or HIF-2α-YC (Figure S2D) and CRY1-YN constructs were applied in the assay. In addition, a clear fluorescent signal could be observed when HIF-1α-YC was coexpressed with ARNT-YN or PER1-YC with CRY1-YN as positive controls (Figures 4E, S2D, and S2E). By contrast, when HIF-1α-YC or HIF-2α -YC was coexpressed with a vector encoding only YN (Figures 4E and 4F) no fluorescence was detected. Likewise, the coexpression of YC and YN did not result in the generation of fluorescence, indicating that the separated non-fluorescent fragments did not spontaneously self-assemble (Figures 4E and 4F). Together, these results indicate that HIF-1α and HIF-2α can interact with CRY1 in living cells. To find out which domain within CRY1 interacts with HIF-1α, we expressed Myc-tagged HIF1α along with different HA-tagged CRY1 protein variants in HEK293 cells (Figure 5A ) and analyzed their interaction by coimmunoprecipitation experiments. Full-length CRY1 interacted with HIF-1α, but this interaction was lost when CRY1 proteins lacking either the CC and tail region or only the tail region were employed in the assays (Figure 5B). Thus, these data indicate that the tail region of CRY1 is necessary for the interaction of CRY1 with HIF-1α. To map the exact HIF-1α domain that interacts with CRY1, we designed several Myc-tagged HIF-1α deletion mutants comprising one or several domains (Figure 6A ) and performed coimmunoprecipitation experiments in HA-CRY1 expressing HEK293 cells. When full-length HIF-1α or its deletion variants were pulled down with anti-Myc-tag antibodies we found that HA-CRY1 coprecipitated full-length HIF-1α and all deletion variants except the HIF-1α mutant lacking the bHLH domain (Figure 6B). Together, these results indicate that CRY1 interacts with the bHLH domain of HIF-1α. As CRY1 interacts with the HIF-1α bHLH domain, which primarily facilitates binding to DNA at target gene promoters, we next analyzed how CRY1 would affect hypoxia and HIF-dependent gene expression. First, we performed a series of functional reporter gene assays in HepG2 cells. Upon cotransfection of these cells with the hypoxia- and HIF-responsive wild-type (WT) human PAI-1 promoter Luc construct (pGL3-hPAI-806/+19) and an expression vector for CRY1 we found that the hypoxia-mediated increase in Luc activity was reduced in the presence of CRY1. This effect was mediated via the hypoxia responsive element (HRE) within the PAI-1 promoter because a construct with a mutated element (pGL3-hPAI-HREm) did not display this effect on Luc activity (Figure 7A ). Vice versa, when we employed MEFs deficient for either CRY1 (ΔCRY1) or CRY2 (ΔCRY2) to measure PAI-1 expression, we found that deficiency of CRY1, but not CRY2, enhanced PAI-1 levels already under normoxia to almost the same levels as under hypoxia (Figures S3A and S3C). Reintroduction of CRY1 (Figures S3D and S3E) rescued the hypoxia-dependent PAI-1 expression indicating that these effects are largely CRY1 specific. In line with the interaction assays, these rescue effects were dependent on the tail region of CRY1 since only full-length CRY1, but not CRY1-Δtail, rescued the hypoxia-dependent induction of PAI-1 (Figures S3D and S3E). Thus, CRY1 appears to function as inhibitor of the hypoxia-dependent PAI-1 gene expression. Next, we aimed to substantiate the inhibitory effect of CRY1 for HIF-driven genes and HIFs. To do this, we used bona fide hypoxia reporters containing either three WT HIF-binding HREs or three mutated HREs in front of the SV40 promoter and the luciferase gene. We then cotransfected these reporters together with a CRY1 expression vector or empty control vector into HepG2 cells. Hypoxia induced Luc activity in the control cells, whereas expression of CRY1 predominantly abolished Luc activity under hypoxia (Figure 7B). This effect was specific because mutation of critical nucleotides in the HRE (pGL3-HREm) abolished the induction of Luc activity under hypoxia and the repressive effect of CRY1 (Figure 7B). As HIFs are dimers consisting of HIF-α and HIF-β (ARNT) subunits, we next investigated whether the CRY1 effect is primarily dependent on each subunit by using the pGL3-HRE Luc construct an" @default.
• W2919465720 created "2019-03-11" @default.
• W2919465720 creator A5003895098 @default.
• W2919465720 creator A5012458079 @default.
• W2919465720 creator A5016682290 @default.
• W2919465720 creator A5030462166 @default.
• W2919465720 creator A5034747590 @default.
• W2919465720 creator A5037856060 @default.
• W2919465720 creator A5039341868 @default.
• W2919465720 creator A5046928222 @default.
• W2919465720 creator A5060356675 @default.
• W2919465720 creator A5066942749 @default.
• W2919465720 creator A5075806156 @default.
• W2919465720 creator A5080518547 @default.
• W2919465720 creator A5081447281 @default.
• W2919465720 creator A5088529716 @default.
• W2919465720 creator A5091080977 @default.
• W2919465720 date "2019-03-01" @default.
• W2919465720 modified "2023-10-17" @default.
• W2919465720 title "The Circadian Clock Protein CRY1 Is a Negative Regulator of HIF-1α" @default.
• W2919465720 cites W114098355 @default.
• W2919465720 cites W1585056952 @default.
• W2919465720 cites W1744822607 @default.
• W2919465720 cites W181093158 @default.
• W2919465720 cites W1918437779 @default.
• W2919465720 cites W1959897390 @default.
• W2919465720 cites W1969515408 @default.
• W2919465720 cites W1969734439 @default.
• W2919465720 cites W1973788573 @default.
• W2919465720 cites W1978248876 @default.
• W2919465720 cites W1986568068 @default.
• W2919465720 cites W1988377666 @default.
• W2919465720 cites W1995475470 @default.
• W2919465720 cites W1996527248 @default.
• W2919465720 cites W1997179110 @default.
• W2919465720 cites W1997364930 @default.
• W2919465720 cites W1997426777 @default.
• W2919465720 cites W1999094582 @default.
• W2919465720 cites W2001528167 @default.
• W2919465720 cites W2004498120 @default.
• W2919465720 cites W2005729255 @default.
• W2919465720 cites W2010575484 @default.
• W2919465720 cites W2013244539 @default.
• W2919465720 cites W2018021093 @default.
• W2919465720 cites W2020898077 @default.
• W2919465720 cites W2026178836 @default.
• W2919465720 cites W2027121745 @default.
• W2919465720 cites W2036061009 @default.
• W2919465720 cites W2036196597 @default.
• W2919465720 cites W2045944658 @default.
• W2919465720 cites W2046084416 @default.
• W2919465720 cites W2046770420 @default.
• W2919465720 cites W2048667125 @default.
• W2919465720 cites W2053210485 @default.
• W2919465720 cites W2054944151 @default.
• W2919465720 cites W2057080091 @default.
• W2919465720 cites W2059474406 @default.
• W2919465720 cites W2061769555 @default.
• W2919465720 cites W2066650294 @default.
• W2919465720 cites W2068628426 @default.
• W2919465720 cites W2068787504 @default.
• W2919465720 cites W2069698188 @default.
• W2919465720 cites W2069742753 @default.
• W2919465720 cites W2084887961 @default.
• W2919465720 cites W2086473844 @default.
• W2919465720 cites W2088348233 @default.
• W2919465720 cites W2091531174 @default.
• W2919465720 cites W2094589586 @default.
• W2919465720 cites W2102054988 @default.
• W2919465720 cites W2113297137 @default.
• W2919465720 cites W2115340622 @default.
• W2919465720 cites W2117719962 @default.
• W2919465720 cites W2123049006 @default.
• W2919465720 cites W2124287509 @default.
• W2919465720 cites W2126618928 @default.
• W2919465720 cites W2129503064 @default.
• W2919465720 cites W2133221833 @default.
• W2919465720 cites W2137694179 @default.
• W2919465720 cites W2142478420 @default.
• W2919465720 cites W2150108640 @default.
• W2919465720 cites W2151599212 @default.
• W2919465720 cites W2164628441 @default.
• W2919465720 cites W2169658668 @default.
• W2919465720 cites W2234675074 @default.
• W2919465720 cites W2293813232 @default.
• W2919465720 cites W2484887837 @default.
• W2919465720 cites W2534086390 @default.
• W2919465720 cites W2536252358 @default.
• W2919465720 cites W2537243438 @default.
• W2919465720 cites W2560007524 @default.
• W2919465720 cites W2563439156 @default.
• W2919465720 cites W2566090297 @default.
• W2919465720 cites W2583671727 @default.
• W2919465720 cites W2602526631 @default.
• W2919465720 cites W2605106690 @default.
• W2919465720 cites W2606681577 @default.
• W2919465720 cites W2624675086 @default.
• W2919465720 cites W2757554288 @default.

• next