Functional analysis of a novel KRAB/C2H2 zinc finger protein Mipu1.
A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C2H2 zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor.
Jiang
Lei
L
Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, PR China.
Tang
Daolin
D
Wang
Kangkai
K
Zhang
Huali
H
Yuan
Can
C
Duan
Dayue
D
Xiao
Xianzhong
X
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology
C486563:BCL11A protein, human; D002352:Carrier Proteins; C520710:Mipu1 protein, rat; D009687:Nuclear Proteins; D012097:Repressor Proteins; D004247:DNA; C486788:Zfp472 protein, mouse