Open AccessAmino acid 1–209 is essential for PDX‐1‐mediated repression of human CMV IE promoter activity
Author(s) -
CHEN Jing,
CHEN Lei,
LI Ge,
CHENG Lu,
HUANG Yin,
ZHANG Jiaxin,
FAN Weiwei,
LU Daru
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00420.x
Subject(s) - psychological repression , electrophoretic mobility shift assay , microbiology and biotechnology , promoter , mutant , reporter gene , promoter activity , endogeny , dna binding domain , chemistry , biology , gene , transcription factor , gene expression , biochemistry
Aim: To explore the different roles of pancreatic duodenal homeobox factors‐1 (PDX‐1) domains in PDX‐1 mediated repression of human cytomegalovirus immediately early (CMV IE) promoter. Methods: A series of truncated PDX‐1 mutants were constructed. The binding of PDX‐1 and CMV IE promoter was identified by electrophoretic mobility shift assay (EMSA). The dual‐reporter assay was applied to examine the repression activities of PDX‐1 mutants on CMV IE promoter. In addition, RNAi technology was used to specifically knock down the endogenous PDX‐1 expression. Results: The reporter assay indicated that compared to the mock controls (pEGFP‐N2), overexpression of PDX‐1 resulted in a 41% decrease of CMV IE promoter activity in the 293 cells ( P <0.05) and 43% decrease in HeLa cells ( P <0.05), and the repression levels of various truncated mutants played on CMV IE promoter were different. Specific knock down of the endogenous PDX‐1 expression significantly restored the activity of CMV IE promoter. EMSA demonstrated that domain 3 is necessary for nuclear localization and DNA binding activity of PDX‐1. However, binding of PDX‐1 alone to CMV IE promoter was not sufficient to inhibit its transcriptional activity, and other domains of PDX‐1 presented were also required. Conclusion: Our data suggested that the DNA binding activity of PDX‐1 domain 3 and the cooperative binding of PDX‐1 domain 1/2 with other proteins were required for PDX‐1 mediated repression of CMV IE promoter.