Research article

Functional characterization of two CITED3 homologs (gcCITED3a and gcCITED3b) in the hypoxia-tolerant grass carp, Ctenopharyngodon idellus

Patrick KS Ng^2* , Sung-Kay Chiu^1* , Theresa FN Kwong¹ , Richard MK Yu³ , Minnie ML Wong¹ and Richard YC Kong¹

¹  Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong Special Administrative Region, PR China

²  Department of Biochemistry, Chinese University of Hong Kong, New Territories, Hong Kong Special Administrative Region, PR China

³  School of Environmental and Life Sciences, University of Newcastle, New South Wales, Australia

author email corresponding author email* Contributed equally

BMC Molecular Biology 2009, 10:101doi:10.1186/1471-2199-10-101

Published:	3 November 2009

Abstract

Background

CITED proteins belong to a family of non-DNA-binding transcriptional co-regulators that are characterized by a conserved ED-rich domain at the C-terminus. This family of genes is involved in the regulation of a variety of transcriptional responses through interactions with the CBP/p300 integrators and various transcription factors. In fish, very little is known about the expression and functions of CITEDs.

Results

We have characterized two closely related but distinct CITED3 genes, gcCited3a and gcCited3b, from the hypoxia-tolerant grass carp. The deduced gcCITED3a and gcCITED3b proteins share 72% amino acid identity, and are highly similar to the CITED3 proteins of both chicken and Xenopus. Northern blot analysis indicates that the mRNA expression of gcCited3a and gcCited3b is strongly induced by hypoxia in the kidney and liver, respectively. Luciferase reporter assays demonstrated that both gene promoters are activated by gcHIF-1. Further, ChIP assays comparing normal and hypoxic conditions reveal differential in vivo binding of gcHIF-1 to both gene promoters in kidney and liver tissues. HRE-luciferase reporter assays demonstrated that both gcCITED3a and gcCITED3b proteins inhibit gcHIF-1 transcriptional activity, and GST pull-down assays confirmed that both proteins bind specifically to the CH1 domain of the grass carp p300 protein.

Conclusion

The grass carp gcCITED3a and gcCITED3b genes are differentially expressed and regulated in different fish organs in response to hypoxic stress. This is the first report demonstrating in vivo regulation of two closely-related CITED3 isogenes by HIF-1, as well as CITED3 regulation of HIF-1 transcriptional activity in fish. Overall, our findings suggest that unique molecular mechanisms operate through these two gcCITED3 isoforms that likely play an important regulatory role in the hypoxic response in the grass carp.