Functional characterization of the regulatory region of human CD2-associated protein promoter in HEK 293 cells.
BACKGROUND
The mRNA of CD2-associated protein (CD2AP) was found to be changed in glomerular diseases. The promoter plays an important role in the regulation of gene expression, but the characterization of the human CD2AP promoter has not been systematically analyzed in HEK 293 cells.


OBJECTIVE
To analyze in detail the promoter of human CD2AP in HEK 293 cells.


METHODS
The transcriptional initiation sites were identified by 5' RACE. Promoter activities were detected by series deletion and mutational luciferase analyses.


RESULTS
Multiple transcriptional start sites were identified. Progressive deletion analysis from both 5' and 3' ends revealed two kinds of promoter activity. One basic promoter activity was located within 500 bp upstream of ATG. Fragments of further upstream 100 bp increased the promoter activity 5-fold. Two Sp1/Sp3 sites were in this region. Mutations of these two sites reduced the transcriptional activity by 50%. Sp1 increased the activity, whereas Sp3 decreased the activity. Deletion of 9 single nucleotide polymorphism sites and 3 Lmx1b sites did not change the transcriptional activity.


CONCLUSIONS
Sp1/Sp3 binding sites play a critical role in the CD2AP regulation. These findings should facilitate studies on the clinical mutational and polymorphism analysis.
Su
Xin-Ming
XM
Department of Pediatrics, The First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
Ren
Wei
W
Lu
Chao
C
Chen
Ji-Qing
JQ
Wu
Sheng-Hua
SH
Chen
Rong-Hua
RH
Zhou
Guo-Ping
GP
D024506:5' Flanking Region; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D009404:Nephrotic Syndrome / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site
D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; D018398:Homeodomain Proteins; C112149:LIM homeobox transcription factor 1 beta; D060850:LIM-Homeodomain Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor