Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene

International Journal of Molecular Sciences
Ruirui ZhangMinggang Lei

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). T...Continue Reading

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Methods Mentioned

BETA
pull
transfection
pull down
ChIP
ChIP-PCR
PCR
Assay
Immunoprecipitation

Software Mentioned

SAS

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