High fidelity screening of regulatory sequences in apolipoprotein(a)-plasminogen cluster.
Identifying networks of gene expression regulation is one of the major tasks in the post-genomic era, this demands firstly high throughput identification of regulatory elements. Apolipoprotein(a)-plasminogen cluster is closely related to both atherosclerosis and thrombosis, and forms a link between the two systems. The mechanism regulating expression of this cluster, through which the balance between atherosclerosis and thrombosis is achieved, is far from been fully understood. Polymerase chain reaction based strategies for screening of regulatory sequences are both simple and efficient yet face severe problems of contamination and sequence mutations. In this work, entity self-competition electrophoresis mobility shift assay and differential library hybridization are introduced to overcome the problems. Cis-element candidates are picked out from a library generated from the screening region, thus avoiding the effect of repetitive amplifications. Sequence fidelity of the selected fragments is guaranteed and one by one identification of each fragment by self-competition is unnecessary. Using this method, 32 potential regulatory elements in apolipoprotein(a)-plasminogen gene cluster were found, including three of the four known DNase I hypersensitive sites in the cluster, attesting the efficiency of our method. We also find an intron-locating element, which is repeatedly screened out. A new in vivo electrophoresis mobility shift assay method based on in situ digestion of crosslinked chromatin DNA and southern hybridization is established for the detection of in vivo protein binding status in the intron region. The result supports our hypothesis that the element may play an important role in expression regulation or evolution of apolipoprotein(a) gene.
Lv
Xiang
X
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing.
Shi
Hong-Zhe
HZ
Liu
De-Pei
DP
Hao
De-Long
DL
Zhang
Shen
S
Xin
Li
L
Li
Xing-Guo
XG
Xu
Hai-Ming
HM
Liang
Chih-Chuan
CC
D016632:Apolipoprotein A-I / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism; D005810:Multigene Family; D010958:Plasminogen / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D010958:Plasminogen