Transcriptional regulation and functional implication of the grass carp CITED1 (gcCITED1) in the negative regulation of HIF-1

Hypoxia triggers a broad range of gene responses that are primarily mediated by the transcription factor, hypoxia-inducible factor-1 (HIF-1) that complexes with the transcriptional coactivator CREB-binding protein/p300 (CBP/p300). In mammals, members of the CBP/p300-interacting transactivators with ED-rich tail (CITED) family, such as CITED2 and CITED4, bind CBP/p300 with high affinity and thereby negatively regulate HIF-1 transactivation. In fish, we have previously shown that two CITED3 homologues from the hypoxia-tolerant grass carp (Ctenopharyngodon idellus) are induced by hypoxia/HIF-1 and able to inhibit HIF-1 transactivation. Here we report the identification and functional characterization of the grass carp CITED1 (gcCITED1) protein as a new repressor of HIF-1-mediated transcriptional activity. Expression of gcCITED1 mRNA was increased in heart, kidney and liver in vivo after exposure to hypoxia. Luciferase reporter and ChIP assays, respectively, indicated the inducibility of the gcCITED1 promoter by gcHIF-1 and the in vivo binding of gcHIF-1 to the gcCITED1 promoter. Ectopic overexpression of gcCITED1 significantly attenuated HIF-1-dependent transactivation of a HRE-luciferase reporter gene. Furthermore, GST pull-down confirmed that gcCITED1 specifically binds via its CR2 domain to the CH1 region of the grass carp p300 coactivator. Overall, our findings suggest that the hypoxia/gcHIF-1-inducible gcCITED1 may function in a negative feedback loop to regulate gcHIF-1 activity in response to hypoxia stress.