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.
Subject(s)
Adaptation, Physiological , Carps/metabolism , Fish Proteins/chemistry , Fish Proteins/metabolism , Hypoxia/metabolism , Sequence Homology, Amino Acid , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Carps/genetics , Chromatin Immunoprecipitation , Cricetinae , Cricetulus , Fish Proteins/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Luciferases/metabolism , Molecular Sequence Data , Phylogeny , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Response Elements/genetics , Time Factors , Transcriptional Activation/genetics , p300-CBP Transcription Factors/metabolismSubject(s)
DNA, Complementary/chemistry , Fish Proteins/metabolism , Oryzias/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Motifs , Animals , Cell Hypoxia , Female , Fish Proteins/chemistry , Fish Proteins/genetics , Male , Molecular Sequence Data , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Receptors, Leptin , Sequence AlignmentABSTRACT
In this study we identified a putative virulence-associated DNA methyltransferase (MTase) gene carried on a novel 22.79-kb pathogenicity island-like element (VPAI) in V. parahaemolyticus. The V. parahaemolyticus MTase gene was shown by PCR to be prevalent (>98%) in pandemic thermostable direct hemolysin gene-positive isolates, which suggests that VPAI may confer unique virulence traits to pandemic strains of V. parahaemolyticus.
