Sw. Ng et al., MOLECULAR-CLONING AND CHARACTERIZATION OF THE PROMOTER FOR THE CHINESE-HAMSTER DNA TOPOISOMERASE II-ALPHA GENE, The Journal of biological chemistry, 270(43), 1995, pp. 25850-25858
To investigate the mechanisms governing the expression of DNA topoisom
erase II alpha, the Chinese hamster topoisomerase II alpha gene has be
en cloned and the promoter region analyzed. There are several transcri
ptional start sites clustered in a region of 30 base pairs, with the m
ajor one being 102 nucleotides upstream from the ATG translation initi
ation site. Sequencing data reveal one GC box and a total of five inve
rted CCAAT elements (ICEs) within a region of 530 base pairs upstream
from the major transcription start site. Sequence comparison between t
he human and Chinese hamster topoisomerase II alpha gene promoter regi
ons shows a high degree of homology centered at the ICEs and GC box. I
n vitro DNase I footprinting results indicate protection by binding pr
oteins at and around each ICE on both DNA strands. However, no obvious
protection was observed for the GC box, Competition gel mobility shif
t assays with oligonucleotides containing either the wild-type or muta
ted ICE sequences suggest that identical or similar proteins specifica
lly bind at each ICE, although with different affinities for individua
l ICE sequences. Chloramphenicol acetyltransferase assays employing ne
sted 5'-deletions of the 5'-flanking sequence of the gene demonstrate
that the sequence between -186 and +102, which contains three proximal
ICEs, is sufficient for near wild-type level of promoter activity. Wh
en these three ICEs were gradually replaced with sequences which do no
t interact with the binding proteins, reducing promoter activity of th
e resulted constructs was observed, In conjunction with results from f
ootprinting and gel mobility shift studies, the transient gene express
ion finding suggests that the ICEs are functionally important for the
transcriptional regulation of the topoisomerase II alpha gene.