Molecular pathogenesis of Gilbert's syndrome: decreased TATA-binding protein binding affinity of UGT1A1 gene promoter.
OBJECTIVE
Gilbert's syndrome is a congenital, nonhemolytic, unconjugated hyperbilirubinemia. The most common genotype of Gilbert's syndrome is the homozygous polymorphism, A(TA)7TAA, in the promoter of the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1), with a thymine adenine insertion in the TATA-box-like sequence, which results in a decrease in UGT1A1 activity. The mechanism responsible for this decrease in UGT1A1 activity, however, has not been elucidated. To clarify the mechanism underlying this deficiency in UGT1A1 activity in patients with Gilbert's syndrome.


METHODS
The promoter activity assay using the wild-type A(TA)6TAA or the mutant A(TA)7TAA promoter and a luciferase reporter was performed in two different hepatoma cell lines. The binding affinity for a nuclear protein complex or for TATA-binding protein was evaluated by a competitive electophoretic mobility shift assay using wild-type or mutant TATA-box-like oligonucleotide probes and nuclear extract or TATA-binding protein. The formation of complexes between TATA-binding protein and wild-type or mutant oligonucleotide probes was also studied by a quantitive electophoretic mobility shift assay.


RESULTS
A TA insertion in the TATA-box-like sequence of the promoter activity of UGT1A1 gene. A competitive electrophoretic mobility shift assay showed a decrease in nuclear protein complex binding affinity and TATA-binding protein binding affinity of the mutant TATA-box-like sequence A(TA)7TAA. When the mutants A(TA)5TAA and A(TA)8TAA were also compared, quantitative electrophoretic mobility shift assay demonstrated that the TATA-binding protein binding affinity progressively decreased as the number of TA repeats in the TATA-box-like sequence increased.


CONCLUSIONS
TA insertion in the TATA-box-like sequence of the UGT1A1 promoter affected its binding affinity for TATA-binding protein, causing a decrease in its activity. This explains the pathogenesis of Gilbert's syndrome.
Hsieh
Tsai-Yuan
TY
Department of Internal Medicine, Tri-Service General Hospital, Taipei, Taiwan, ROC.
Shiu
Tzu-Yue
TY
Huang
Shih-Ming
SM
Lin
Hsuan-Hwai
HH
Lee
Tai-Chi
TC
Chen
Peng-Jen
PJ
Chu
Heng-Cheng
HC
Chang
Wei-Kuo
WK
Jeng
King-Song
KS
Lai
Michael M C
MM
Chao
You-Chen
YC
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005878:Gilbert Disease / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016254:Mutagenesis, Insertional; D010597:Pharmacogenetics; D011401:Promoter Regions, Genetic; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D000099148:UGT1A1 Enzyme
D017931:DNA Primers; D014453:Glucuronosyltransferase; D035181:TATA-Box Binding Protein; D000099148:UGT1A1 Enzyme