Next, we unraveled the mechanism by which HBXIP activates Lin28B promoter. We used DNA fragment of −1199/−1073 nt region of Lin28B promoter as the probe. EMSA indicated that there was an interaction between nuclear proteins and the probe (Fig. 4a, lane 3). While, a supershift band was observed when HBXIP antibody was added (Fig. 4a, lane 4), suggesting that HBXIP is able to bind to the −1199/−1073 nt region of Lin28B promoter. However, the purified HBXIP protein alone failed to bind to the −1199/−1073 nt fragment in vitro (Supporting Information Fig. S2a), implying that HBXIP indirectly interacts with Lin28B promoter in the cells. To explore the interaction site between HBXIP and Lin28B promoter, we decomposed the −1199/−1073 nt region into five overlapping DNA fragments, and the overlapping region contains eight nucleotides between the adjacent two fragments (Supporting Information Fig. S2b). Finally, EMSA assay identified that the DNA segment covering −1178/−1148 nt region of Lin28B promoter was responsible for interacting with HBXIP (Supporting Information Fig. S2b). Next, using promoter analysis program (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/prom omenu.cgi?dirDB=TF_8.3&Option=3&idCon=128523136200), we predicted the putative recognition motifs of three transcription factors with high scores, such as GATA-1, TF II D, and GR-β, in the −1178/-1148 nt region of Lin28B promoter. EMSA assay showed that the interaction between nuclear extracts of MCF-7 cells and the probe of −1178/-1148 promoter region of Lin28B was disrupted when the binding site of TF II D was mutated in the promoter. However, the mutant binding site of the GATA-1 or GR-β failed to affect the interaction (Fig. 4b), suggesting that TF II D is responsible for the interaction between the nuclear extracts and Lin28B promoter. Additionally, the luciferase activities of Lin28B promoter were markedly decreased when the TF II D binding site was mutated in the cells (Fig. 4c). Thus, we speculated that HBXIP might activate the Lin28B promoter through interacting with TF II D. To test this speculation, we investigated whether HBXIP could bind to TF II D. Because the transcription factor TF II D is a complex composed of the TBP and TAFs, and the TBP component recognizes and binds to the TATA-box to initiate assembly of the transcription complex.[25, 26] Thus, to elucidate the relationship between HBXIP and TF II D, we focused on the relationship between HBXIP and TBP as a substitute of TF II D. Next, the Co-IP assays were performed and the results revealed that HBXIP and TBP could bind to each other in MCF-7 cells (Fig. 4d, left panel). To further verify whether the combination between HBXIP and TBP is direct or not, GST pull-down assays were performed. The protein of TBP and HBXIP was expressed, respectively, in bacteria. We found that the TBP protein with His-tag could be pulled down by GST-HBXIP, but not by the control GST alone (Fig. 4d, right panel), suggesting that HBXIP directly binds to TBP protein in the cells. These data suggested that HBXIP directly bound to TF II D. Next, we asked whether the promoter activity of Lin28B is dependent on the interaction between HBXIP and TBP. Luciferase reporter gene assays showed that over-expression of HBXIP or TBP resulted in the enhanced activity of the core promoter, while the core promoter activity was decreased when HBXIP or TBP was knocked down by HBXIP siRNA-2 or TBP siRNA, respectively (Fig. 4e). Importantly, we also found that the elevated activities of Lin28B promoter mediated by ectopic HBXIP (or TBP) could be abolished by the TBP siRNA (or HBXIP siRNA-2) in MCF-7 cells (Fig. 4e). Moreover, EMSA assay showed that there was a weaker band when TBP antibody was added (Supporting Information Fig. S2c, lane 3) compared to the controls containing no antibodies (Supporting Information Fig. S2c, lane 2). These data strongly demonstrate that the promoter activity of Lin28B is dependent on the interaction between HBXIP and TBP, suggesting that HBXIP functions as a co-activator of transcription factor TF II D. Taken together, we conclude that HBXIP, as a co-activator of TF II D, stimulates Lin28B promoter through directly binding to TBP subunit of TF II D.
Figure 4. HBXIP stimulates Lin28B promoter via directly binding to TBP subunit of TF II D. (a) The interaction between Lin28B promoter (−1199/−1073 nt) probe and HBXIP was tested by EMSA. (b) The interaction between nuclear proteins and Lin28B promoter (−1178/−1148 nt) probe with the mutant binding sites of GATA-1, TF II D or GR-β was detected, respectively, by EMSA. (c) The luciferase activities of the Lin28B core promoter with wild type (or mutant binding sites of TF II D) were, respectively, determined by luciferase reporter gene assays in MCF-7 cells transfected with pCMV-HBXIP (80 ng). *p < 0.05, **p hx003C 0.01, Student's t-test. All experiments were performed at least three times. (d) The interaction between HBXIP and TBP was detected by Co-IP assays in MCF-7 cells in vivo. The direct interaction between recombinant GST-HBXIP and His-TBP proteins was detected by GST pull-down assay and western blot analysis in vitro. (e) The luciferase activities of Lin28B core promoter (−1199/−1073nt) were detected by luciferase reporter gene assays in MCF-7 cells transiently transfected with HBXIP siRNA-2 (70 nM) (or TBP siRNA), pCMV-HBXIP (100 ng) (or pCMV-TBP), pCMV-HBXIP (100 ng)/TBP siRNA (70 nM) (or pCMV-TBP/HBXIP siRNA-2), respectively.
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