BMC Mol Biol
November 3, 2009;
Functional characterization of two CITED3 homologs (gcCITED3a and gcCITED3b) in the hypoxia-tolerant grass carp, Ctenopharyngodon idellus.
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
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
, 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
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. 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
BMC Mol Biol
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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.
Isolation and expression of a novel member of the CITED family.