CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.     

Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp

We have isolated a 1586-bp full-length CITED3 cDNA from grass carp which specifies for a cAMP-responsive element-binding protein/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain protein. The cDNA, designated as gcCITED3, has an open reading frame of 762 bp and encodes a protein of 253 amino acids with a predicted molecular mass of 28.3 kDa and pI of 6.4. Pairwise comparison showed that gcCITED3 shares high sequence identity with the CITED3 of zebrafish (94%), chicken (72%) and Xenopus (59%). Northern blot analysis indicated that gcCITED3 is most highly expressed and responsive to hypoxia in the carp kidney. Hypoxic induction was also observed in heart, albeit at a lower level. This is the first report on the isolation of a hypoxia-responsive CITED3 gene from fish.     

CITED2 controls the hypoxic signaling by snatching p300 from the two distinct activation domains of HIF-1α

HIF-1α plays a central role in cellular adaptation to hypoxia, and is closely related to the pathogeneses of life-threatening disorders. HIF-1α induces the expressions of numerous hypoxia-induced genes through two transactivation domains; N-terminal TAD (NAD) and C-terminal TAD (CAD). Furthermore, p300 is known to boost CAD-dependent transactivation, and CBP/p300-interacting transactivator with an ED-rich tail 2 (CITED2) inhibits HIF-1α-driven gene expression by interfering with the interaction between CAD and p300. However, few researches have focused on the role of CITED2 in the regulation of NAD activity, and thus, we addressed this point. CITED2 was found to attenuate the hypoxic activations of NAD-dependent and CAD-dependent genes, suggesting that CITED2 negatively regulates both CAD and NAD. Immunoprecipitation analyses showed that NAD interacts with the Cystein/Histidine region (CH) 1 and CH3 domains of p300. Moreover, CH1 and CH3 both were required for NAD-dependent transactivation. Furthermore, CITED2 was found to inactivate NAD by interfering with NAD binding to CH1, but not to CH3. These results indicate that CITED2 inactivates HIF-1α by blocking p300 recruitment by both NAD and CAD. We also found that pVHL inhibits NAD activity regardless of NAD degradation by blocking the interaction between p300 and NAD. Summarizing, NAD was activated by binding to p300, and this was blocked by either CITED2 or pVHL. We propose that pVHL controls NAD during normoxia and that CITED2 controls NAD during hypoxia. Our results provide a new strategy for controlling HIF-1α.     

CITED2-mediated regulation of MMP-1 and MMP-13 in human chondrocytes under flow shear

CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) is a member of the Cited family of nuclear regulators, previously known as mrg1 (melanocyte-specific gene-related gene 1). CITED2 is inducible by varying stimuli including lipopolysaccharide, hypoxia, and cytokines such as interleukin 9 and interferon gamma. Using the immortalized human chondrocyte cell line, C-28/I2, we investigated whether CITED2 could be responsive to mechanical stimuli, and if so, whether CITED2 could mediate shear-driven regulation of matrix metalloproteinase (MMP) genes. The C-28/I2 cells were cultured under flow shear at 1-20 dyn/cm2, and the role of CIT-ED2 in regulation of MMPs was examined using the plasmids encoding sense and antisense CITED2 DNA sequences. The results showed that flow shear at 5 dyn/cm2 increased CITED2 mRNA and protein levels and down-regulated MMP-1 and MMP-13 mRNA and protein levels as well as enzyme activities. Consistent with the coordinated expression patterns of CITED2 and MMPs, overexpression of CITED2 repressed MMP-1 and MMP-13 mRNA levels and activities, whereas antisense CITED2 plasmids prevented the shear-induced down-regulation of MMP expression. Interleukin-1beta induced the formation of p300-Ets-1 complexes without affecting expression of CITED2. Transforming growth factor-beta as well as flow shear at 5 dyn/cm2 stimulated not only the expression of CITED2 but also the association of CIT-ED2 with p300 by dissociating Ets-1 from p300. These results indicate that CITED2 plays a major role in shear-induced down-regulation of MMP-1 and MMP-13 via a transforming growth factor-beta-dependent pathway.