Cloning and functional analysis of 5'-upstream region of the Pokemon gene.
Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expression of the Pokemon gene in humans, its 5'-upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG-U and NEG-D elements were involved in negative regulation of the Pokemon promoter, whereas the POS-D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG-U, NEG-D and POS-D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG-U and NEG-D elements led to negative regulation of the Pokemon promoter. Moreover, the NEG-U and NEG-D elements needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy.
Yang
Yutao
Y
Laboratory of Protein Engineering, Beijing Institute of Biotechnology, Beijing, China.
Zhou
Xiaowei
X
Zhu
Xudong
X
Zhang
Chuanfu
C
Yang
Zhixin
Z
Xu
Long
L
Huang
Peitang
P
D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; C485514:ZBTB7A protein, human