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Xiaofeng Ding,
Fangliang Zhou,
Fangmei Wang,
Zijian Yang,
Chang Zhou, Jianlin Zhou,
Bo Zhang,
Junmei Yang,
Guangwei Wang,
Zheng Wei,
Xiang Hu,
Shuanglin Xiang,
Jian Zhang
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ABSTRACT: Eps8 was initially identified as a substrate of the epidermal growth factor receptor. Overexpression of Eps8 leads to increased mitogenic signaling and malignant transformation. However, little is known concerning the importance of Eps8 in human gliomas. In this study, we found that Eps8 was overexpressed in 56.6% of human gliomas (WHO grades III and IV) compared with adjacent normal brain tissues by immunohistochemical analysis. The U251 human glioma cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced U251 glioma cell growth and survival by cell survival, MTT and liquid colony formation assays. By contrast, the lentiviral expression of Eps8 siRNA in SHG-44 cells resulted in a significant reduction in cellular growth and proliferation. Furthermore, Eps8 modulated the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), phosphorylated serine-threonine protein kinase Akt and β-catenin expression in glioma cell lines and tissues. These results suggest that Eps8 is overexpressed in human gliomas, and affects glioma cell growth possibly by regulating ERK and Akt/β-catenin signaling. Therefore, Eps8 may represent a novel potential target in human glioma therapy.
Oncology Reports 11/2012; · 1.84 Impact Factor
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ABSTRACT: The broad-complex, tramtrack, and bric-a-brac/poxvirus and zinc finger domain-containing protein tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) was first identified as a gene whose expression can be induced by the tumor necrosis factor alpha. Some studies showed that TNFAIP1 may function in DNA replication, apoptosis and human diseases. However, the definite functions and the mechanisms of TNFAIP1 are poorly known. In this study, we performed a yeast two-hybrid assay and used TNFAIP1 as the bait to screen human brain cDNA library. Potassium channel tetramerisation domain containing 10 (KCTD10) was identified as TNFAIP1-interacting partner. The KCTD10-TNFAIP1 interaction was then confirmed by the in vitro GST pull-down assays and the in vivo co-immunoprecipitation and colocalization assays. In addition, protein degradation and ubiquitin assays revealed TNFAIP1 overexpression resulted in ubiquitin-mediated degradation of KCTD10 proteins, which was significantly alleviated with the proteasome inhibitor MG132 treatment. Furthermore, transient transfection assays with two reporters showed that TNFAIP1 and KCTD10 inhibited the transcriptional activities of nuclear factor kappa B (NF-κB) and activating protein-1 reporters. Taken together, our results indicated the novel interaction and function between KCTD10 and TNFAIP1 in human PDIP1 family.
Molecular Biology Reports 07/2012; 39(11):9911-9. · 2.93 Impact Factor
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ABSTRACT: Transcription factor AP-2α involves in the process of mammalian embryonic development and tumorigenesis. Many studies have shown that AP-2α functions in association with other interacting proteins. In a two-hybrid screening, the regulatory subunit β of protein casein kinase 2 (CK2β) was identified as an interacting protein of AP-2α; we confirmed this interaction using in-vitro GST pull-down and in-vivo co-immunoprecipitation assays; in an endogenous co-immunoprecipitation experiment, we further found the catalytic subunit α of protein casein kinase 2 (CK2α) also exists in the complex. Phosphorylation analysis revealed that AP-2α was phosphorylated by CK2 kinase majorly at the site of Ser429, and such phosphorylation could be blocked by CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in a dose-dependent manner. Luciferase assays demonstrated that both CK2α and CK2β enhanced the transcription activity of AP-2α; moreover, CK2β increased the stability of AP-2α. Our data suggest a novel cellular function of CK-2 as a transcriptional co-activator of AP-2α.
BMB reports 07/2011; 44(7):490-5. · 1.72 Impact Factor
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ABSTRACT: The αB-crystallin (CRYAB) is a member of the small heat shock protein family that can be induced by various stresses and pathological conditions. Aberrant expression of CRYAB has been shown to be associated with several neurological diseases and malignant neoplasms. To identify transcriptional regulators of CRYAB expression, we examined its promoter for binding sites of transcription factors and identified four potential AP-2 binding sites in addition to a p53 binding site reported previously. Although the CRYAB promoter contains four consensus binding sequences of AP-2 and can be activated by AP-2α either in the presence or absence of p53, the luciferase assay showed that AP-2β alone does not regulate the activity of the CRYAB promoter in the absence of p53. However, in the presence of p53, AP-2β can significantly increase the luciferase activities of both the CRYAB promoter and reporter vector pp53-TA-luc, which contains a p53-responsive element, but no AP-2 binding sites. These data suggest that AP-2β enhances transactivation of p53 and regulates CRYAB transcription via p53. Further study demonstrated that AP-2β interacts with p53 and augments its protein stability. Taken together, our results indicate that AP-2β up-regulates the transcription of the CRYAB gene through stabilizing p53.
Molecular Biology Reports 05/2011; 39(1):209-14. · 2.93 Impact Factor
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ABSTRACT: The transcription factor AP-2α functions as a tumor suppressor by regulating various genes that are involved in cell proliferation and apoptosis. Chemotherapeutic drugs including cisplatin induce post-transcriptionally endogenous AP-2α, which contributes to chemosensitivity by enhancing therapy-induced apoptosis. microRNAs (miRNAs) miR-200b, miR-200c and miR-429 (miR-200b/200c/429) are up-regulated in endometrial and esophageal cancers, and their overexpression correlates with resistance to cisplatin treatment. Using computational programs, we predicted that the 3' untranslated region (UTR) of AP-2α gene contains a potential miRNA response element (MRE) for the miR-200b/200c/429 family, and the single nucleotide polymorphism (SNP) site rs1045385 (A or C allele) resided within the predicted MRE. Luciferase assays and Western blot analysis demonstrated that the miR-200b/200c/429 family recognized the MRE in the 3' UTR of AP-2α gene and negatively regulated the expression of endogenous AP-2α proteins. SNP rs1045385 A>C variation enhanced AP-2α expression by disrupting the binding of the miR-200b/200c/429 family to the 3' UTR of AP-2α. The effects of the two polymorphic variants on cisplatin sensitivity were determined by clonogenic assay. The overexpression of AP-2α with mutant 3' UTR (C allele) in the endometrial cancer cell line HEC-1A, which has high levels of endogenous miR-200b/200c/429 and low levels of AP-2α protein, significantly increased cisplatin sensitivity, but overexpression of A allele of AP-2α has no significant effects, compared with mock transfection. We concluded that miR-200b/200c/429 induced cisplatin resistance by repressing AP-2α expression in endometrial cancer cells. The SNP (rs1045385) A>C variation decreased the binding of miR-200b/200c/429 to the 3' UTR of AP-2α, which upregulated AP-2α protein expression and increased cisplatin sensitivity. Our results suggest that SNP (rs1045385) may be a potential prognostic marker for cisplatin treatment.
PLoS ONE 01/2011; 6(12):e29043. · 4.09 Impact Factor
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Jianlin Zhou,
Xi Qiao,
Ling Xiao,
Wei Sun,
Lin Wang,
Hong Li,
Yuan Wu,
Xiaofeng Ding,
Xiang Hu,
Chang Zhou,
Jian Zhang
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ABSTRACT: The putative CCDC106 protein was previously identified as a p53-interacting partner by automated yeast two-hybrid screening, but its sequence and function have not been validated experimentally. Here, we identified three variant transcripts of the CCDC106 gene; these transcripts differ in their second exons due to the use of different splice acceptor site, but encode an identical protein of 280 residues. A bipartite nuclear localisation signal at residues 151-164 mediates the nuclear localisation of CCDC106. Double immunofluorescence staining revealed the colocalisation of endogenous CCDC106 and p53 protein in nuclei. The in vivo interaction between CCDC106 and p53 was confirmed by a co-immunoprecipitation assay. Furthermore, we demonstrated that CCDC106 promotes the degradation of p53 protein and inhibits its transactivity.
FEBS letters 02/2010; 584(6):1085-90. · 3.54 Impact Factor
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ABSTRACT: TNFAIP1 is a protein which can be induced by tumor necrosis factoralpha (TNFalpha) and interleukin-6 (IL-6), it may play roles in DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in these physiological processes. In this paper, CK2beta was identified as a partner of TNFAIP1 by screening the HeLa cDNA library in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested that the phosphorylation of TNFAIP1 may be required for its functions.
Molecular Biology Reports 10/2009; 37(6):2967-73. · 2.93 Impact Factor
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ABSTRACT: The mouse maelstrom (MAEL) gene has been found to be expressed in male germ cells and to play a role in spermatogenesis. Here, we cloned the human MAEL gene by digital differential display and found that, among human tissues, MAEL is only expressed in the testis, but it is also expressed in various cancer cell lines. The transcription start site of the MAEL gene is 74-bp upstream of the start codon. The region from -216 to +150 is the basal promoter of the MAEL gene, and a CpG island (-295 to +148) is located in this region. Treatment with the demethylating agent 5'-Aza-2-Deoxycytidine significantly upregulated MAEL expression. These results suggest that MAEL is a novel cancer/testis-associated gene and is regulated by DNA methylation.
Molecular Biology Reports 09/2009; 37(5):2355-60. · 2.93 Impact Factor
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Hong Li,
Qian Liu,
Xiang Hu,
Du Feng,
Shuanglin Xiang,
Zhicheng He,
Xingwang Hu, Jianlin Zhou,
Xiaofeng Ding,
Chang Zhou,
Jian Zhang
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ABSTRACT: Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) signaling, and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR). However, the details of how human ZCCHC12 involve in the NS-XLMR still remain unclear. In this study, we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization. Multiple-tissue northern blot analysis indicated that ZCCHC12 is highly expressed in human brain. Furthermore, in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain, midbrain, and diencephalon regions of mouse E10.5 embryos. Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcriptional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a coactivator. In conclusion, we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.
Acta Biochimica et Biophysica Sinica 08/2009; 41(7):535-44. · 1.38 Impact Factor
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Mingjun Liu,
Zhenhua Sun,
Aidong Zhou,
Hong Li,
Liping Yang,
Chang Zhou,
Rushi Liu,
Xiang Hu, Jianlin Zhou,
Shuanglin Xiang,
Jian Zhang
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ABSTRACT: Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an immediate-early response gene of endothelium induced by TNF alpha. However, little is really known concerning the TNFAIP1 expression regulation. To better understand how TNFAIP1 expression is regulated, we functionally characterized the promoter region of human TNFAIP1 gene. Deletion mutation analysis, gel electrophoretic mobility shift, and site-directed mutagenesis assays allowed the identification of one functional Sp1-binding site within the human TNFAIP1 core promoter region. Moreover, chromatin immunoprecipitation analysis indicated that Sp1 was associated in vivo with the TNFAIP1 promoter. Further, Sp1 overexpression enhanced TNFAIP1 promoter activity. These findings suggest that Sp1 is implicated in the control of basal TNFAIP1 gene expression. Accordingly, Sp1 is supposed to be involved in the elevation of TNFAIP1 in response to TNF alpha induction, and thus participate in inflammation-associated angiogenesis.
Molecular Biology Reports 08/2009; 37(4):1699-705. · 2.93 Impact Factor
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Rushi Liu,
Aidong Zhou,
Daolong Ren,
Ailan He,
Xiang Hu,
Wenfeng Zhang,
Liping Yang,
Mingjun Liu,
Hong Li, Jianlin Zhou,
Shuanglin Xiang,
Jian Zhang
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ABSTRACT: Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase delta, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2alpha could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2alpha indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2alpha to this region showed opposite effects.
FEBS Journal 02/2009; 276(4):1114-24. · 3.79 Impact Factor
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Rushi Liu,
Aidong Zhou,
Daolong Ren,
Ailan He,
Xiang Hu,
Wenfeng Zhang,
Liping Yang,
Mingjun Liu,
Hong Li, Jianlin Zhou,
Shuanglin Xiang,
Jian Zhang
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ABSTRACT: Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase δ, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5′-flanking region (−609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from −108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2α could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2α indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2α to this region showed opposite effects.
FEBS Journal 01/2009; 276(4):1114 - 1124. · 3.79 Impact Factor
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Xiaofeng Ding,
Chang Luo, Jianlin Zhou,
Yingli Zhong,
Xiang Hu,
Fangliang Zhou,
Kaiqun Ren,
Lu Gan,
Ailan He,
Jiaolian Zhu,
Xiang Gao,
Jian Zhang
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ABSTRACT: AP-2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP-2alpha-interacting partners, a yeast two-hybrid screen was performed in human brain cDNA library. One of the identified clones encodes potassium channel tetramerization domain-containing 1 (KCTD1). We demonstrated the novel KCTD1-AP-2alpha interaction in vitro by GST pull-down assays and in vivo by co-immunoprecipitation assays and mapped the interaction domains to the N-termini of both proteins. In addition, we observed that the two proteins were completely co-localized in the nuclei of mammalian cells. Transient transfection assays using four promoters containing AP-2-binding sites confirmed that KCTD1 significantly repressed AP-2alpha-mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1-mediated repression of AP-2alpha transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP-2alpha, suggesting that KCTD1 specifically acts as a negative regulator of AP-2alpha. Finally, we found that KCTD1 interacted with three major members of the AP-2 family and inhibited their transcriptional activities. Taken together, our results indicate the novel function of KCTD1 as the transcriptional repressor for AP-2 family, especially for AP-2alpha.
Journal of Cellular Biochemistry 01/2009; 106(2):285-95. · 2.87 Impact Factor
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ABSTRACT: The human TESTIN (TES) is a putative tumor suppressor and localizes to the cytoplasm as a component of focal adhesions and cell contacts. TES contains a PET domain in the NH(2)-terminus and three tandem LIM domains in the COOH-terminus. It has been hypothesized that interactions between two termini of TES might lead to a "closed" conformational state of the protein. Here, we provide evidence for different conformational states of TES. We confirmed that the NH(2)-terminus of TES can interact with its third LIM domain in the COOH-terminus by GST pull-down assays. In addition, antisera against the full-length or two truncations of TES were prepared to examine the relationship between the conformation and cellular distribution of the protein. We found that these antisera recognize different regions of TES and showed that TES is co-localised with the marker protein B23 in nucleolus, in addition to its localization in endoplasmic reticulum (ER). Furthermore, our co-immunoprecipitation (co-IP) analysis of TES and B23 demonstrated their co-existence in the same complex. Taken together, our results suggest that TES has different conformational states in different cellular compartments, and a "closed" conformational state of TES may be involved in nucleolar localization.
Molecular and Cellular Biochemistry 09/2008; 320(1-2):85-92. · 2.06 Impact Factor
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ABSTRACT: The CCHC-type zinc finger motif has numerous biological activities (such as DNA binding and RNA binding) and can also mediate protein-protein interaction. This article gives a primary report about the human ZCCHC9 gene. Protein ZCCHC9 contains four CCHC motifs and is highly conserved in humans, mice, and rats. The whole cDNA sequence of the ZCCHC9 gene has been amplified by PCR and a number of plasmids have been constructed for further study. The results show that ZCCHC9 is localized in the nucleus, and especially concentrated in the nucleolus. It is highly expressed in the brain and testicles of the mouse. This has been confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). In situ hybridization of the mouse brain indicates that ZCCHC9 is mainly expressed in the cerebral cortex. Reporter gene assay shows that ZCCHC9 suppresses the transcription activities of NF-kappa B and SRE, and may play roles in the Mitogen-Activated Protein Kinase (MAPK) signaling transduction pathway.
Journal of Genetics and Genomics 09/2008; 35(8):467-72. · 1.88 Impact Factor
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Xiaofeng Ding,
Changzheng Fan, Jianlin Zhou,
Yingli Zhong,
Rushi Liu,
Kaiqun Ren,
Xiang Hu,
Chang Luo,
Shunyong Xiao,
Yeqi Wang,
Du Feng,
Jian Zhang
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ABSTRACT: Transcription factor AP-2 regulates transcription of a number of genes involving mammalian development, differentiation and carcinogenesis. Recent studies have shown that interaction partners can modulate the transcriptional activity of AP-2 over the downstream targets. In this study, we reported the identification of GAS41 as an interaction partner of AP-2beta. We documented the interaction both in vivo by co-immunoprecipitation as well as in vitro through glutathione S-transferase (GST) pull-down assays. We also showed that the two proteins are co-localized in the nuclei of mammalian cells. We further mapped the interaction domains between the two proteins to the C-termini of both AP-2beta and GAS41, respectively. Furthermore, we have identified three critical residues of GAS41 that are important for the interaction between the two proteins. In addition, by transient co-expression experiments using reporter containing three AP-2 consensus binding sites in the promoter region, we found that GAS41 stimulates the transcriptional activity of AP-2beta over the reporter. Finally, electrophoretic mobility shift assay (EMSA) suggested that GAS41 enhances the DNA-binding activity of AP-2beta. Our data provide evidence for a novel cellular function of GAS41 as a transcriptional co-activator for AP-2beta.
Nucleic Acids Research 02/2006; 34(9):2570-8. · 8.03 Impact Factor
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ABSTRACT: Rat potassium channel tetramerisation domain-containing 10 (KCTD10) gene was cloned and identified as a novel member of polymerase delta-interacting protein 1 (PDIP1) gene family. Rat KCTD10 is highly expressed in lung and moderately expressed in heart and testis. KCTD10 shares significant similarity in amino acid sequence to PDIP1 and can interact with the small subunit of DNA polymerase delta and PCNA as PDIP1 does. Like PDIP1, the expression of KCTD10 gene can be induced by TNF-alpha in NIH3T3 cells.
Biochimica et Biophysica Acta 08/2005; 1729(3):200-3. · 4.66 Impact Factor
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ABSTRACT: The mouse polymerase delta-interacting protein 1 gene, PDIP1, is mapped to chromosome 7F3 region, spans approximately 16.7kb, and is organized into six exons. The transcription start site (TSS) was determined to be G, corresponding to position of 162-bp upstream of the translation start codon. The promoter region was found to lack TATA box or CCAAT box, instead, a CpG island was detected surrounding TSS. The region from -162 to +114 is required for basal transcriptional regulation of mouse PDIP1 gene, contains two AP-2 and two Sp1 binding sites. The Sp1 site upstream of TSS activates, while the other Sp1 site and two AP-2 sites suppress the transcription activity of mouse PDIP1 gene.
FEBS Letters 04/2005; 579(7):1715-22. · 3.54 Impact Factor
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ABSTRACT: Human polymerase delta-interacting protein 1 (PDIP1) is a tumor necrosis factor alpha and interleukin 6 inducible protein that interacts directly with proliferating cell nuclear antigen (PCNA) and the small subunit (p50) of DNA polymerase delta. PDIP1 binds PCNA and p50 simultaneously and stimulates polymerase delta activity in vitro in the presence, but not the absence, of PCNA. It has been suggested that PDIP1 provides a link between cytokine activation and DNA replication in eukaryotes. Here these authors report the cloning of two rat genes homologous to human PDIP1, termed rat PDIP1 and rat tumor necrosis factor-induced protein 1 (TNFAIP1). The rat PDIP1 is mapped to chromosome 1q36 cM region, spans approximately 18.7 kb, and is organized into six exons. The rat TNFAIP1 gene is mapped to chromosome 10q25 cM, spans approximately 12.9 kb, and is composed of seven exons. The deduced proteins of rat PDIP1 and rat TNFAIP1 share 63.1% sequence identity with each other and are highly conserved in the majority of the middle portion of the two proteins, which encode a BTB/POZ domain at the N-terminus and a PCNA binding motif (QTKV-EFP) at the C-terminus, respectively. The BTB / POZ domain and the PCNA binding motif are highly conserved during the evolution. Both rat PDIP1 and rat TNFAIP1 were demonstrated to interact with PCNA via BIAcore, GST pull-down, and co-immunoprecipitation assays. Like the human PDIP1, both rat PDIP1 and rat TNFAIP1 stimulate polymerase delta activity in vitro in a PCNA-dependent way.
Journal of Experimental Zoology Part A Comparative Experimental Biology 04/2005; 303(3):227-40.
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Xiaofeng Ding,
Changzheng Fan, Jianlin Zhou,
Yingli Zhong,
Rushi Liu,
Kaiqun Ren,
Xiang Hu,
Chang Luo,
Shunyong Xiao,
Yeqi Wang,
Du Feng,
Jian Zhang
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[hide abstract]
ABSTRACT: Transcription factor AP-2 regulates transcription of a number of genes involving mammalian development, differentiation and carcinogenesis. Recent studies have shown that interaction partners can modulate the transcriptional activity of AP-2 over the downstream targets. In this study, we reported the identification of GAS41 as an interaction partner of AP-2β. We documented the interaction both in vivo by co-immunoprecipitation as well as in vitro through glutathione S -transferase (GST) pull-down assays. We also showed that the two proteins are co-localized in the nuclei of mammalian cells. We further mapped the interaction domains between the two proteins to the C-termini of both AP-2β and GAS41, respectively. Furthermore, we have identified three critical residues of GAS41 that are important for the interaction between the two proteins. In addition, by transient co-expression experiments using reporter containing three AP-2 consensus binding sites in the promoter region, we found that GAS41 stimulates the transcriptional activity of AP-2β over the reporter. Finally, electrophoretic mobility shift assay (EMSA) suggested that GAS41 enhances the DNA-binding activity of AP-2β. Our data provide evidence for a novel cellular function of GAS41 as a transcriptional co-activator for AP-2β.
