Aquatic hypoxia is a stress commonly encountered by many aquatic organisms, including fish, in both seawater and freshwater habitats. To cope with hypoxic stress, fish have developed a variety of adaptive strategies including increasing oxygen delivery and anaerobic energy production as well as reduction in energy expenditure. In mammals, a number of physiological and biochemical responses to hypoxia are modulated by the hypoxia-inducible factor-1 (HIF-1) transcription factor which acts as a master regulator of dozens of hypoxia-responsive genes. Among these, the human CITED2 gene has been shown to negatively regulate HIF-1 transcriptional activity. So far, most of our knowledge about CITED proteins and their functions is derived from mammalian systems and very little is known in fish. This study aims to investigate the biological roles of different CITED proteins and their effects on HIF-1 activity in the grass carp (Ctenopharyngodon idella), in order to better understand the underlying mechanisms of hypoxia responses in fish. In this study, full-length or near full-length cDNAs encoding four different CITED proteins - gcCITED1 (1855 bp), gcCITED2 (1935 bp) gcCITED3a (1586 bp) and gcCITED3b (3061 bp) - have been isolated from the grass carp using degenerate RT-PCR and RACE PCR techniques. The open reading frames of gcCITED1, gcCITED2, gcCITED3a and gcCITED3b are 432 bp, 669 bp, 750 bp and 723 bp in length and encode proteins of 143, 222, 249 and 240 amino acid residues, respectively. All four gcCITED proteins contain the family signature domain CR2, as well as a CR1 domain, whereas the CR3 domain is conserved only in the gcCITED2, 3a and 3b proteins. Pair-wise sequence comparison indicated that gcCITED3a and gcCITED3b share high sequence identity (68%) with each other and low sequence identity with gcCITED1 and gcCITED2 (21% - 37%). Moreover, pair-wise comparison between the gcCITED proteins and CITED proteins from various vertebrate species showed that gcCITED1 (44% - 94%), gcCITED2 (64% - 75%), gcCITED3a (78% - 93%) and gcCITED3b (68% - 95%) share high sequence similarity with their homologs in other vertebrates. Homology search of various vertebrate and invertebrate genome databases showed that while CITED1, CITED2, CITED3a and CITED3b are also present in the zebrafish (Danio rerio) genome, only CITED1, CITED3a and CITED3b isoforms were detected in the Fugu and Japanese medaka (Oryzias latipes) genomes. In addition, bioinformatic analysis showed that the human genome contains only CITED1, CITED2 and CITED4, whereas CITED3a- and CITED3b-like homologs were not found. CITED-like genes were also not found in the genomes of invertebrates such as fruit fly (Drosophila melanogaster), mosquito (Anopheles gambiae), nematode (Caenorhabditis elegans) and purple sea urchin (Strongylocentrotus purpuratus). Northern blot analysis of normoxic and hypoxic grass carp tissues revealed that gcCITED1, gcCITED3a and gcCITED3b are differentially expressed under normoxia and hypoxia. The highest expression of gcCITED1, gcCITED3a and gcCITED3b mRNAs under normoxia was detected in heart, kidney and liver, respectively, while hypoxic induction of gcCITED1 was detected in heart, kidney and liver; induction of gcCITED3a was detected in kidney, gill, heart and liver; and induction of gcCITED3b was observed in all tissues examined with the exception of muscle. The promoters of gcCITED1, gcCITED3a and gcCITED3b were analyzed using luciferase reporter and chromatin immunoprecipitation (ChIP) assays, and basal promoter activity of gcCITED3b was the highest, followed by gcCITED1 and gcCITED3a. Promoter activities of all three gcCITED genes were induced by overexpression of gcHIF-1 (from 1.36- to 1.79-fold). ChIP assays demonstrated that hypoxia induced differential patterns of in vivo gcHIF-1α-binding to various promoter regions of the three gcCITED genes in liver and kidney of grass carp. The effect of gcCITED ectopic expression on HIF-1 transcriptional activity was examined by gene transfection in CHO cells and the findings indicated that each of the three gcCITED proteins has the capacity (to varying degree) to inhibit gcHIF-1 transactivation activity, and that the inhibitory effect of the gcCITED proteins is mediated by the highly conserved CR2 domain. Physical interaction between the gcCITED1, gcCITED3a and gcCITED3b proteins and the endogenous CBP/p300 of CHO cells was examined by co-immunoprecipitation and the results showed that the wild-type and CR2 deletion mutants of gcCITED3a and gcCITED3b are able to interact with the CBP/p300 in CHO cells. These indicated that the CH1 domain of p300 may not be the only domain that interacts with the gcCITED proteins and that other ancillary proteins may be involved in vivo. In contrast, no apparent interaction was observed between the wild-type or CR2 deletion mutant of gcCITED1 and CBP/p300. GST pull-down assays confirmed that the CR2 domains of gcCITED3a and gcCITED3b physically interact with the CH1 domain of p300. Overall, the findings suggest that all three examined gcCITED genes are induced under hypoxia, probably due to gcHIF-1 binding to the HRE(s) in their promoters, and the gcCITED proteins inhibit gcHIF-1 transactivation activity via competition for the CH1 domain in CBP/p300 co-activators.
| Date of Award | 2 Oct 2007 |
|---|
| Original language | English |
|---|
| Awarding Institution | - City University of Hong Kong
|
|---|
| Supervisor | Yuen Chong Richard KONG (Supervisor) |
|---|