GC-rich promoter elements maximally confers estrogen-induced transactivation of LRP16 gene through ERalpha/Sp1 interaction in MCF-7 cells.
LRP16 gene has been characterized as an estrogen-responsive gene. One 1/2ERE/GC-rich site was previously identified to be indispensable for -676/-214 (region A) fragment within LRP16 regulatory region to confer E2 action. Here, we report that -213/-24 fragment (region B) has higher E2-responsiveness than that of region A in MCF-7 cells, but not in HeLa cells. Deletion and mutation analyses of region B showed that multiple GC-sites are involved in the E2-stimulated response and one 30-bp fragment (-213 to -184 bp) is essential for conferring maximum E2-responsiveness. Results from the cotransfection assays containing Sp1-siRNA revealed that Sp1 is required for the basal transcription activity and E2-responsiveness of both regions A and B. Northern blot analysis demonstrated that inhibition of Sp1 in MCF-7 cells not only decreased the basal expression of LRP16, but markedly impaired its upregulation by E2. Results from gel mobility shift assays exhibited the direct binding of Sp1 protein to the 28-bp fragment (-211 to -184 bp), which was enhanced by the ERalpha titer. Moreover, the functional interaction of ERalpha and Sp1 proteins in the presence of E2 at the GC-rich sites in region B was confirmed by chromatin immunoprecipitation (ChIP) assays. In general, these results demonstrate that GC-rich sites in the proximal promoter of LRP16 gene are sufficient for E2 activation of LRP16 and the -213/-184 fragment containing only one GC site is essential for the maximal induction in MCF-7 cells. We also provide a model for Sp1-dependent regulation of genes by E2 through GC-rich motifs.
Han
Wei Dong
WD
Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing 100853, PR China. hanwdrsw69@yahoo.com
Si
Yi Ling
YL
Zhao
Ya Li
YL
Li
Qi
Q
Wu
Zhi Qiang
ZQ
Hao
Hao Jie
HJ
Song
Hai Jing
HJ
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D005260:Female; D020862:GC Rich Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects
D004273:DNA, Neoplasm; D047628:Estrogen Receptor alpha; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D004958:Estradiol; D002265:Carboxylic Ester Hydrolases; C476832:MACROD1 protein, human