Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp

We have isolated a 1586-bp full-length CITED3 cDNA from grass carp which specifies for a cAMP-responsive element-binding protein/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain protein. The cDNA, designated as gcCITED3, has an open reading frame of 762 bp and encodes a protein of 253 amino acids with a predicted molecular mass of 28.3 kDa and pI of 6.4. Pairwise comparison showed that gcCITED3 shares high sequence identity with the CITED3 of zebrafish (94%), chicken (72%) and Xenopus (59%). Northern blot analysis indicated that gcCITED3 is most highly expressed and responsive to hypoxia in the carp kidney. Hypoxic induction was also observed in heart, albeit at a lower level. This is the first report on the isolation of a hypoxia-responsive CITED3 gene from fish.     

CITED2 controls the hypoxic signaling by snatching p300 from the two distinct activation domains of HIF-1α

HIF-1α plays a central role in cellular adaptation to hypoxia, and is closely related to the pathogeneses of life-threatening disorders. HIF-1α induces the expressions of numerous hypoxia-induced genes through two transactivation domains; N-terminal TAD (NAD) and C-terminal TAD (CAD). Furthermore, p300 is known to boost CAD-dependent transactivation, and CBP/p300-interacting transactivator with an ED-rich tail 2 (CITED2) inhibits HIF-1α-driven gene expression by interfering with the interaction between CAD and p300. However, few researches have focused on the role of CITED2 in the regulation of NAD activity, and thus, we addressed this point. CITED2 was found to attenuate the hypoxic activations of NAD-dependent and CAD-dependent genes, suggesting that CITED2 negatively regulates both CAD and NAD. Immunoprecipitation analyses showed that NAD interacts with the Cystein/Histidine region (CH) 1 and CH3 domains of p300. Moreover, CH1 and CH3 both were required for NAD-dependent transactivation. Furthermore, CITED2 was found to inactivate NAD by interfering with NAD binding to CH1, but not to CH3. These results indicate that CITED2 inactivates HIF-1α by blocking p300 recruitment by both NAD and CAD. We also found that pVHL inhibits NAD activity regardless of NAD degradation by blocking the interaction between p300 and NAD. Summarizing, NAD was activated by binding to p300, and this was blocked by either CITED2 or pVHL. We propose that pVHL controls NAD during normoxia and that CITED2 controls NAD during hypoxia. Our results provide a new strategy for controlling HIF-1α.