[Mapping of FHL2 transcription activation domain].
FHL2, a member of LIM-only protein family, plays an important role in transcription regulation, apoptosis, cancer development and progression. In this study, a mammalian transcription activation system was constructed by using DNA binding domain(DBD) of GAL4 and luciferase reporter gene with DBD binding sequence, and used for mapping of FHL2 transcription activation domain. First, the coding sequence of GAL4-DBD was inserted into expression vector pcDNA3, generating the pDBD recombinant plasmid, then the wild-type FHL2 and its mutants were fused in-frame with GAL4-DBD, resulting in expression vectors for wild-type FHL2 and its mutants. All of the recombinant plasmids were transfected into 293T cells. Western blot assay showed that all of the fusion proteins were expressed. The analysis of FHL2 transcription activation properties by using the GAL4-luciferase reporter gene indicated that wild-type FHL2 had activation activity in both 293T and MCF-7 cells. The deletion of the half LIM domain at the N-terminus severely impaired the capacity of FHL2 to stimulate transcription. The mutant lacking the LIM domain at the C-terminus was totally inactive, while the deletion of two LIM domains at the C-terminus partially recovered its ability to stimulate transcription. The deletion of the second LIM domain at C-terminus did not alter the activation capacity of FHL2. These results suggest that the last LIM domain at the C-terminus of FHL2 is critical for its transcription activation function, the second LIM domain at the C-terminus may be a negative regulation region, but this negative regulation depends on the last LIM domain. Mapping of transcription activation domain of FHL2 lays solid basis for further study on various FHL2 functions.
Yan
Jing-Hua
JH
Beijing Institute of Biotechnology, Beijing 100850, China.
Ye
Qi-Nong
QN
Fang
Yan
Y
Zhu
Jian-Hua
JH
Huang
Cui-Fen
CF
D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D010957:Plasmids; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015533:Transcriptional Activation; D014162:Transfection
C105987:FHL2 protein, human; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D014157:Transcription Factors; D004247:DNA