Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
BMC Mol Biol
2009 Nov 03;10:101. doi: 10.1186/1471-2199-10-101.
Show Gene links
Show Anatomy links
Functional characterization of two CITED3 homologs (gcCITED3a and gcCITED3b) in the hypoxia-tolerant grass carp, Ctenopharyngodon idellus.
Ng PK
,
Chiu SK
,
Kwong TF
,
Yu RM
,
Wong MM
,
Kong RY
.
???displayArticle.abstract???
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. 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 carpp300 protein. 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.
Figure 1. Amino acid sequence alignment of gcCITED3a and gcCITED3b with related homologs from other fish species. Amino acid residues in the CR1, CR2 and CR3 conserved domains typically found in CITED proteins are boxed and labeled. Amino acid residues in the CR2 domain that interact with the CH1 domain of CBP/p300 are indicated with a plus (+). The QH-rich domain of fish CITED3a/CITED3b is highlighted by a grey overline. The leucine-rich nuclear export signal (Leu-rich NES) is indicated above the alignment with a parenthesis. olCITED3a and olCITED3b, Oryzias latipes CITED3a and CITED3b; omCITED3a and omCITED3b, Oryzias melastigma CITED3a and CITED3b; fCITED3a and fCITED3b, Fugu CITED3a and CITED3b; gcCITED3a and gcCITED3b, grass carp CITED3a and CITED3b; zCITED3a and zCITED3b, zebrafish CITED3a and CITED3b; xlCITED3, Xenopus CITED3; chCITED, chicken CITED3.
Figure 2. Phylogeny of fish gcCITED3a and gcCITED3b proteins. The tree was constructed by the neighbor-joining method of the MEGA v3.1 program using fish CITED1 proteins as outgroup. The bootstrap support for each branch (1000 replications) is shown. The branch lengths are proportional to the number of substitutions between sequences. The GenBank/EMBL/Swissprot accession numbers of the CITED sequences used are given in Table 1.
Figure 3. Northern blot analysis of gcCited3a and gcCited3b. Fish tissues (brain, gill, heart, kidney, liver, and muscle) were isolated from normoxic (N) and hypoxic (H) grass carp following 4 h and 96 h of exposure as indicated. Total RNA from each tissue was isolated and analyzed by Northern hybridization using gene-specific probes for gcCITED3a and gcCITED3b. Ethidium bromide-stained 28S rRNA is shown as the loading control. A representative Northern blot derived from the tissues of one normoxic and one hypoxic fish (from a total of three in each group) is shown here.
Figure 4. Transcriptional activation of gcCited3a and gcCited3b gene promoters and identification of gcHIF-1 binding sites by ChIP-PCR. CHO cells were transiently transfected with the pCITED3a(-1817/+30), pCITED3b(-1713/+76) or pGL3-Basic luciferase vectors together with pCMV-gcHIF1α, pCMV-gcARNT2b (HIF-1 complex) and pSVβ-gal plasmid or the empty pCMV vector. Luciferase was normalized against β-gal activity and data represent the means ± S.D. of three independent experiments. Asterisks indicate significant differences, P ≤ 0.05, between pCITED3a and pCITED3b promoter constructs and the pGL3-Basic vector.
Figure 5. ChIP-PCR analysis of gcHIF-1 binding to the gcCited3a and gcCited3b gene promoters. A. The primer-sets (indicated by opposing arrows of the same grey scale) for ChIP-PCR amplification of different regions of the 5'-flanking sequence of gcCITED3a are shown, and the regions analyzed are demarcated on the right panel. B. The primer-sets (indicated by opposing arrows of the same grey scale) for ChIP-PCR amplification of different regions of the 5'-flanking sequence of gcCITED3b are shown, and the regions analyzed are demarcated on the right panel. Representative ChIP results from kidney (upper panel) and liver (lower panel) of normoxic (left panel) and hypoxic (right panel) fish. The PCR products of samples without immunoprecipitation (Input), immunoprecipitated with anti-gcHIF-1α anti-serum (IP) and water (-ve) were resolved by gel electrophoresis. A PCR band appears if a DNA region is bound by gcHIF-1 in the IP (immunoprecipitated) samples.
Figure 6. Transactivation of HRE-luciferase activity by gcHIF-1 is inhibited by gcCITED3a and gcCITED3b. CHO cells were co-transfected with a pSV40-EpoHRE-luciferase reporter and pSV-β-gal vector (control for transfection efficiency) along with pBK-CMV-gcHIF-1α and pBK-CMV-gcARNT2b or empty pCMV-TNT vector. Wild-type pCMV-gcCITED3a or deletion mutant plasmid pCMV-gcCITED3aΔCR2 (panel A); or pCMV-gcCITED3b or deletion mutant plasmid pCMV-gcCITED3bΔCR2 (panel B) was also co-transfected with pBK-CMV-gcHIF-1α. Relative luciferase activity is the ratio of luciferase over β-galactosidase activity. Transfections were performed in triplicates and data are expressed as means ± S.D. of three independent experiments. Single asterisks (*) indicate significant differences between CHO cells co-transfected with (filled bars) or without (open bars) gcHIF-1 in the absence of gcCITED, p < 0.05; double asterisks (**) indicate significant differences in luciferase activities of CHO cells co-transfected with pCMV-gcCITED or pCMV-gcCITEDΔCR2 in the presence of gcHIF-1, p < 0.05.
Figure 7. gcCITED3a and gcCITED3b physically interact with CBP/p300. A. Immunoprecipitation and Western blot analysis. CHO cells were separately transfected with pGFP-gcCITED3a, pGFP-gcCITED3aΔCR2, pGFP-CITED3b, or pGFP-CITED3bΔCR2, and whole cell lysates prepared and immunoprecipitated with anti-CBP antibody (Santa Cruz). The immunoprecipitates were resolved on 12% denaturing SDS-PAGE and Western blot analysis was carried out to detect GFP fusion proteins using anti-GFP antibody (Santa Cruz). B. GST pull-down assays. GST protein only or GST protein fused to gc-p300/CH1 (CH1 domain of grass carp p300) was purified from bacterial culture and immobilized on glutathione-Sepharose beads. In-vitro-transcribed and -translated [35S]-methionine-labeled gcCITED3 or gcCITED3ΔCR2 was incubated with purified GST-fused CH1 or GST alone as indicated in the figure.
Andrews,
Isolation and expression of a novel member of the CITED family.
2000, Pubmed
Andrews,
Isolation and expression of a novel member of the CITED family.
2000,
Pubmed
Bamforth,
Cited2 controls left-right patterning and heart development through a Nodal-Pitx2c pathway.
2004,
Pubmed
Bamforth,
Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator.
2001,
Pubmed
Benita,
An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia.
2009,
Pubmed
Bhattacharya,
ExCITED about HIF.
2003,
Pubmed
Bhattacharya,
Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1.
1999,
Pubmed
Bragança,
Human CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED) 4, a new member of the CITED family, functions as a co-activator for transcription factor AP-2.
2002,
Pubmed
Chou,
Cited2 modulates TGF-beta-mediated upregulation of MMP9.
2006,
Pubmed
Ema,
Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300.
1999,
Pubmed
Fox,
CITED4 inhibits hypoxia-activated transcription in cancer cells, and its cytoplasmic location in breast cancer is associated with elevated expression of tumor cell hypoxia-inducible factor 1alpha.
2004,
Pubmed
Freedman,
Structural basis for negative regulation of hypoxia-inducible factor-1alpha by CITED2.
2003,
Pubmed
Gawantka,
Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning.
1998,
Pubmed
,
Xenbase
Glenn,
MRG1 binds to the LIM domain of Lhx2 and may function as a coactivator to stimulate glycoprotein hormone alpha-subunit gene expression.
1999,
Pubmed
Haggerty,
A strategy for identifying transcription factor binding sites reveals two classes of genomic c-Myc target sites.
2003,
Pubmed
Ivan,
HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.
2001,
Pubmed
Law,
Cloning and expression analysis of two distinct HIF-alpha isoforms--gcHIF-1alpha and gcHIF-4alpha--from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus.
2006,
Pubmed
Lisy,
Turn me on: regulating HIF transcriptional activity.
2008,
Pubmed
Mole,
Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts.
2009,
Pubmed
Ng,
Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp.
2003,
Pubmed
,
Xenbase
Nikinmaa,
Oxygen-dependent gene expression in fishes.
2005,
Pubmed
Ohh,
Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein.
2000,
Pubmed
Plisov,
Cited1 is a bifunctional transcriptional cofactor that regulates early nephronic patterning.
2005,
Pubmed
,
Xenbase
Rees,
Structure and sequence conservation of a putative hypoxia response element in the lactate dehydrogenase-B gene of Fundulus.
2001,
Pubmed
Rees,
A novel hypoxia-response element in the lactate dehydrogenase-B gene of the killifish Fundulus heteroclitus.
2009,
Pubmed
Rissanen,
Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a poikilothermic vertebrate, crucian carp (Carassius carassius).
2006,
Pubmed
Semenza,
Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene.
1991,
Pubmed
Shi,
The transcriptional activity of CITED1 is regulated by phosphorylation in a cell cycle-dependent manner.
2006,
Pubmed
Stroka,
HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia.
2001,
Pubmed
Sun,
MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity.
1998,
Pubmed
Tien,
Identification of the CREB-binding protein/p300-interacting protein CITED2 as a peroxisome proliferator-activated receptor alpha coregulator.
2004,
Pubmed
Wang,
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
1995,
Pubmed
Webster,
The effect of hypoxia in development.
2007,
Pubmed
Yahata,
Cloning of mouse Cited4, a member of the CITED family p300/CBP-binding transcriptional coactivators: induced expression in mammary epithelial cells.
2002,
Pubmed
Yahata,
The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, enhancing their functional link to the Smad transcription factors.
2000,
Pubmed
Yahata,
Selective coactivation of estrogen-dependent transcription by CITED1 CBP/p300-binding protein.
2001,
Pubmed
Yin,
The essential role of Cited2, a negative regulator for HIF-1alpha, in heart development and neurulation.
2002,
Pubmed
Yokota,
CITED2-mediated regulation of MMP-1 and MMP-13 in human chondrocytes under flow shear.
2003,
Pubmed
Zhang,
Isolation, characterization and expression analysis of a hypoxia-responsive glucose transporter gene from the grass carp, Ctenopharyngodon idellus.
2003,
Pubmed
