An alternative form of nucleolysin binds to a T-cluster DNA in the silencer element of platelet factor 4 gene.
The cDNA of a T-cluster binding protein (TCBP) has been cloned using the Southwestern method. The cDNA sequence of TCBP reveals that it has 78% homology to that of nucleolysin, a factor involved in apoptosis in cytolytic lymphocyte target cells. In particular, the 0.8kb sequences of the 5'-half of both cDNAs were identical. However, nucleolysin has a lysosome-targeting motif at the carboxy terminus, while TCBP has a hydrophobic sequence instead. Southern blot experiments have revealed that both cDNA sequences existed in the same YAC clone derived from chromosome 10. This strongly suggests that the TCBP cDNA is an alternatively spliced product of the nucleolysin/TCBP gene. The TCBP mRNA is ubiquitously expressed, not only in megakaryocytic cells but also in other hematopoietic cells. However, when HEL cells were induced to differentiate to megakaryocytes by DMSO, TCBP mRNA was reduced, while PF4 gene expression increased simultaneously. Gel mobility shift analysis demonstrated that recombinant TCBP specifically bound to the T-cluster and the proximal T-rich region of the PF4 promoter. Co-transfection experiments showed that TCBP reduced the gene expression from the PF4 promoter. On the other hand, TCBP did not affect expression from the PF4 promoter in which the T-cluster and the T-rich region had been removed. These results indicate that TCBP may participate in the regulation of PF4 gene expression by binding to the T-cluster and the T-rich sequence.
Doi
T
T
Faculty of Pharmaceutical Sciences, Osaka University, Suita, Japan.
Minami
T
T
Itoh
M
M
Aburatani
H
H
Kawabe
Y
Y
Kodama
T
T
Kondo
N
N
Satoh
Y
Y
Asayama
T
T
Imanishi
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D003012:Microbial Collagenase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010978:Platelet Factor 4 / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D010978:Platelet Factor 4; D003012:Microbial Collagenase