Amino acid 1-209 is essential for PDX-1-mediated repression of human CMV IE promoter activity.
OBJECTIVE
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.


CONCLUSIONS
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.
Chen
Jing
J
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Chen
Lei
L
Li
Ge
G
Cheng
Lu
L
Huang
Yin
Y
Zhang
Jia-xin
JX
Fan
Wei-wei
WW
Lu
Da-ru
DR
D001483:Base Sequence; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics*; D005801:Genes, Homeobox; D005822:Genetic Vectors; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics*; D034622:RNA Interference; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D018398:Homeodomain Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein