Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene

Abstract

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5'-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from -604 to -554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from -604 to -532 bp) and may affect the process of myogenesis.

Keywords: ROCK1; Sp1; regulation; transcription factor.

Figure 1

5’-Deletion analysis of the porcine ROCK1 promoter activity. Schematic representation of the progressive deletions of porcine ROCK1 5’-flanking region in pGL3-Basic vector and the relative activities of ROCK1 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.

Figure 2

Site-directed mutation of Sp1 binding sites in ROCK1-P5 fragment. (A) Schematic structure of site-directed mutagenesis in the putative Sp1 binding sites (the black slash) of porcine ROCK1 gene. LUC represents the Luciferase gene in the vectors; (B,C) Luciferase activity of site-directed mutagenesis in PK and C2C12 cells. Statistical differences of relative activities were analyzed in the same cells; ** p < 0.01, data were expressed as means ± SD of three replicates.

Figure 3

Binding of Sp1 with the ROCK1-P5 fragment was analyzed in vitro and in vivo. The first (A), the second (B) and the third (C) 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; (D) The three probes were incubated with PK NE, respectively; (E) Proteins of PK extracted from DNA-pull down materials were detected by Western blot. The total non-denaturing proteins/Streptavidin MagneSphere^® 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 (D,E). The competitor/competitor-mutant probes were 50-fold excess and arrows indicated the specific DNA-protein complex bands; (F) Schematic diagram of the Sp1 binding sites in the porcine ROCK1-P5 fragment; (G) ChIP assay of Sp1 binding to porcine ROCK1-P5 fragment in PK cells. The in vivo interaction of Sp1 and Sp3 with porcine ROCK1 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.

Figure 4

Sp1 stimulates the expression of porcine ROCK1 gene. (A) Over-expression of Sp1 up-regulated ROCK1 luciferase activity; (B) Over-expression efficacy of Sp1; (C,D) Over-expression of Sp1 stimulated ROCK1 expression at mRNA and protein level; (E) The interferences efficacy of siRNA; (F) Suppressing Sp1 reduced the ROCK1 promoter activity; (G,H) Inhibition of Sp1 suppressed ROCK1 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. * p < 0.05, ** p < 0.01.

Figure 5

Sp1 promotes the process of myogenesis. Over-expression of Sp1 significantly stimulated MyoD, MyoG, MyHC mRNA expression in C2C12 cells, ** p < 0.01.