Truncation and mutagenesis analysis of the human X-arrestin gene promoter.
X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.
Fujimaki
Takuro
T
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, 1638 N.W. 10th Avenue, Miami, FL 33136, USA.
Huang
Zhen-Yong
ZY
Kitagawa
Hitoshi
H
Sakuma
Hitoshi
H
Murakami
Akira
A
Kanai
Atsushi
A
McLaren
Margaret J
MJ
Inana
George
G
D019390:Arrestins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012160:Retina / Q000378:metabolism; D012175:Retinoblastoma / Q000235:genetics / Q000473:pathology; D017384:Sequence Deletion; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D019390:Arrestins; D011993:Recombinant Fusion Proteins; C082333:arrestin3; D001616:beta-Galactosidase