Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene
<p>5’-Deletion analysis of the porcine <span class="html-italic">ROCK1</span> promoter activity. Schematic representation of the progressive deletions of porcine <span class="html-italic">ROCK1</span> 5’-flanking region in pGL3-Basic vector and the relative activities of <span class="html-italic">ROCK1</span> promoter corresponding to the progressive deletions. The predicted transcription start site (TSS, the red arrow in the figure) was set +1, differs from the TSS in NCBI database. The pGL3-control/basic vectors were used as positive/negative control, while pRL-TK was used as internal control. Data were expressed as means ± SD of three replicates.</p> "> Figure 2
<p>Site-directed mutation of Sp1 binding sites in <span class="html-italic">ROCK1</span>-P5 fragment. (<b>A</b>) Schematic structure of site-directed mutagenesis in the putative Sp1 binding sites (the black slash) of porcine <span class="html-italic">ROCK1</span> gene. LUC represents the <span class="html-italic">Luciferase</span> gene in the vectors; (<b>B</b>,<b>C</b>) Luciferase activity of site-directed mutagenesis in PK and C2C12 cells. Statistical differences of relative activities were analyzed in the same cells; ** <span class="html-italic">p</span> < 0.01, data were expressed as means ± SD of three replicates.</p> "> Figure 3
<p>Binding of Sp1 with the <span class="html-italic">ROCK1</span>-P5 fragment was analyzed <span class="html-italic">in vitro</span> and <span class="html-italic">in vivo</span>. The first (<b>A</b>), the second (<b>B</b>) and the third (<b>C</b>) biotin-labeled probes were incubated with the NE of PK cells. Lane 1 was the negative control without NE; the reagents were incubated in the absence competitor probes in Lane 2 or in presence of 50× excess competitor (Lane 3)/competitor-mutant (Lane 4) probes, respectively; (<b>D</b>) The three probes were incubated with PK NE, respectively; (<b>E</b>) Proteins of PK extracted from DNA-pull down materials were detected by Western blot. The total non-denaturing proteins/Streptavidin MagneSphere<sup>®</sup> Paramagnetic Particles were taken as positive/negative control (PC/NC). The three potential Sp1 binding sites were named as Sp1.1, Sp1.2, and Sp1.3 in (<b>D</b>,<b>E</b>). The competitor/competitor-mutant probes were 50-fold excess and arrows indicated the specific DNA-protein complex bands; (<b>F</b>) Schematic diagram of the Sp1 binding sites in the porcine <span class="html-italic">ROCK1</span>-P5 fragment; (<b>G</b>) ChIP assay of Sp1 binding to porcine <span class="html-italic">ROCK1</span>-P5 fragment in PK cells. The <span class="html-italic">in vivo</span> interaction of Sp1 and Sp3 with porcine <span class="html-italic">ROCK1</span> promoter was determined by ChIP assay, in which Normal mouse IgG was used as negative control. DNA isolated from immunoprecipitated materials was used for PCR amplification, whereas total chromatin was used as input (positive control). The antibodies used in ChIP assay were listed in the right of the figure and the corresponding amplification product obtained here was 107 bp.</p> "> Figure 4
<p>Sp1 stimulates the expression of porcine <span class="html-italic">ROCK1</span> gene. (<b>A</b>) Over-expression of <span class="html-italic">Sp1</span> up-regulated <span class="html-italic">ROCK1</span> luciferase activity; (<b>B</b>) Over-expression efficacy of Sp1; (<b>C</b>,<b>D</b>) Over-expression of Sp1 stimulated <span class="html-italic">ROCK1</span> expression at mRNA and protein level; (<b>E</b>) The interferences efficacy of siRNA; (<b>F</b>) Suppressing <span class="html-italic">Sp1</span> reduced the <span class="html-italic">ROCK1</span> promoter activity; (<b>G</b>,<b>H</b>) Inhibition of <span class="html-italic">Sp1</span> suppressed <span class="html-italic">ROCK1</span> expression at mRNA and protein level. The amount of plasmid was kept constant by the addition of pcDNA3.1 (+) vector. The data were obtained both in PK and C2C12 cells and expressed as means ± SD of three replicates. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 5
<p><span class="html-italic">Sp1</span> promotes the process of myogenesis. Over-expression of <span class="html-italic">Sp1</span> significantly stimulated <span class="html-italic">MyoD</span>, <span class="html-italic">MyoG</span>, <span class="html-italic">MyHC</span> mRNA expression in C2C12 cells, ** <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Identification of the Promoter Region and Regulatory Elements of Porcine ROCK1 Gene.
2.2. The Importance of Sp1 Binding Sites in Porcine ROCK1 Promoter
2.3. Sp1 Binds to the Porcine ROCK1 Promoter in Vitro and in Vivo
2.4. Sp1 Stimulates ROCK1 Gene Expression
2.5. Sp1 Stimulates the Process of Myogenesis
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. In Silico Sequence Analysis
4.3. Rapid Amplification of 5’cDNA Ends (5’-RACE)
4.4. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
4.5. Plasmids’ Construction, Cell Culture, Transfection and Analysis
4.6. Electrophoretic Mobility Shift Assay (EMSA)
4.7. Chromatin Immunoprecipitation (ChIP) Assay
4.8. DNA Pull down Assay
4.9. RNA Interference
4.10. Western Blotting
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Zhang, R.; Feng, X.; Zhan, M.; Huang, C.; Chen, K.; Tang, X.; Kang, T.; Xiong, Y.; Lei, M. Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene. Int. J. Mol. Sci. 2016, 17, 112. https://doi.org/10.3390/ijms17010112
Zhang R, Feng X, Zhan M, Huang C, Chen K, Tang X, Kang T, Xiong Y, Lei M. Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene. International Journal of Molecular Sciences. 2016; 17(1):112. https://doi.org/10.3390/ijms17010112
Chicago/Turabian StyleZhang, Ruirui, Xiaoting Feng, Mengsi Zhan, Cong Huang, Kun Chen, Xiaoyin Tang, Tingting Kang, Yuanzhu Xiong, and Minggang Lei. 2016. "Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene" International Journal of Molecular Sciences 17, no. 1: 112. https://doi.org/10.3390/ijms17010112