A circadian clock gene, PER2, activates HIF‐1 as an effector molecule for recruitment of HIF‐1α to promoter regions of its downstream genes

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor functioning in cellular adaptive responses to hypoxia. Recent studies have suggested that HIF-1 activity is upregulated by one of the important circadian clock genes, period circadian clock 2 (PER2); however, its underlying mechanism remains unclear. Here, we show that PER2 functions as an effector protein for the recruitment of HIF-1a to its cognate enhancer sequence, the hypoxia-response element (HRE). We found that the forced expression of PER2 enhanced HIF-1 activity without influencing expression levels of the regulatory subunit of HIF-1, HIF-1a, at either mRNA or protein levels. A series of coimmunoprecipitation-based experiments revealed that PER2 interacted with HIF-1a and facilitated the recruitment of HIF-1a to HRE derived from vascular endothelial growth factor (VEGF) promoter. The PER2-mediated activation of HIF-1 was observed only when the asparagine residue at position 803 of HIF-1a (HIF-1a N803) was kept unhydroxylated by hypoxic stimulation, by introducing an N803A point mutation, or by an inhibitor of N803-dioxygenase, deferoxamine. However, the extent of PER-2-HIF-1a interaction was equivalent regardless of the N803 hydroxylation status. Taken together, these results suggest that, with the help of an unknown sensor molecule for the N803 hydroxylation status, PER2 functions as an effector molecule for the recruitment of HIF-1 to promoter regions of its downstream genes. Our findings reveal a novel regulatory step in the activation of HIF-1, which can be targeted to develop therapeutic strategies against HIF-1-related diseases, such as cancers.Abbreviations 5 0 -UTR, 5 0 untranslated region; a.a., amino acid; CBP, CREB-binding protein; CDS, coding sequence; DFO, deferoxamine; FIH-1, factorinhibiting HIF-1; Gal4 DBD, Gal4 DNA-binding domain; HDAC, histone deacetylase; HIF-1, hypoxia-inducible factor 1; HRE, hypoxia-response element; IDH3, isocitrate dehydrogenase 3; LY6E, lymphocyte antigen 6 complex, locus E; mTOR, mammalian target of rapamycin; ODD, oxygen-dependent degradation domain; PER2, period circadian clock 2; PHDs, prolyl-4-hydroxylases; PI3K, phosphatidylinositol 3 kinase; PKC, protein kinase C; pVHL, VHL protein; qRT-PCR, quantitative real-time reverse transcriptase PCR; TAD, transactivation domain; UCHL1, ubiquitin C-terminal hydrolase L1; VEGF, vascular endothelial growth factor; VHL, von-Hippel Lindau.

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

“…Hypoxic microenvironment, a common feature in cancer, can activate HIF‐1α and HIF‐1β and the overexpression of these transcription factors contribute to the disturbance in the expression of circadian genes in HCC cells . Recent studies have also suggested that PER2 may function as an effector molecule for the recruitment of HIF‐1 to promoter regions of its downstream genes . Our results indicate that, in addition to the increase in Per1 and 2 expression, melatonin reduces the overexpressed levels of Hif‐1α and Hif‐2α in DEN‐treated mice, thus further supporting their relationship with the circadian clock.…”

“…32 Recent studies have also suggested that PER2 may function as an effector molecule for the recruitment of HIF-1 to promoter regions of its downstream genes. 53 Our results indicate that, in addition to the increase in Per1 and 2 expression, melatonin reduces the overexpressed levels of Hif-1α and Hif-2α in DEN-treated mice, thus further supporting their relationship with the circadian clock.…”

Interplay between environmentally modulated feedback loops – hypoxia and circadian rhythms – two sides of the same coin?

“…PER2 was only able to enhance HIF‐1 activity under hypoxic conditions, suggesting an involvement of FIH‐1. Overexpression of PER2 upregulated HIF‐1 activity only when the target site of FIH‐1 (N803 of HIF1α) was unhydroxylated . However, further analyses revealed that an additional factor must be involved because the direct interaction between HIF‐1α and PER2 was not affected by the hydroxylation status of N803.…”

“…In this issue of The FEBS Journal , Harada et al . studied the interplay between two feedback loops sensing such parameters .…”

“…These facts defined the starting point of Harada et al ., who studied the interplay between the two feedback loops . They focused on the role of period 2 (PER2) in HIF‐1 transactivation, taking into account different recent studies depicting PER2 as a positive modulator of HIF‐1 activity and direct recruitment of HIF‐1 to the PER2 promoter .…”