Analysis of a repressor region in the human neuropeptide Y gene that binds Oct-1 and Pbx-1 in GT1-7 neurons.
The mechanisms dictating the developmental expression of individual neuropeptides within the hypothalamus have not yet been elucidated. In this paper we have studied the cis-acting elements involved in the repression of neuropeptide Y (NPY) gene expression in a gonadotropin-releasing hormone (GnRH) neuronal cell model, GT1-7 cells. Using transient transfection of the human NPY 5(') regulatory region into the GT1-7 neurons, we have found a repressor region located between -867 and -1078. DNase I footprint analysis of this region revealed three specific protein binding elements. Further analysis of the region between -942 and -922bp using electrophoretic mobility shift assays revealed that four different transcription factor-DNA complexes form with GT1-7 nuclear proteins, whereas only three complexes are detected using baby hamster kidney (BHK) cell nuclear extract. Mutation of the consensus binding sequence abolishes all complex formation on the -924/-922 oligonucleotide. Antibody supershift assays revealed that Oct-1 and Pbx-1 antibodies were able to eliminate the appearance of two specific complexes. Therefore we suggest that this region may be important for transcriptional repression of the NPY gene in a heterologous cell model, through complex, coordinate protein-protein interactions.
Mayer
Christopher M
CM
Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King's College Circle, Toronto, Ont., Canada M5S 1A8.
Cai
Fang
F
Cui
Hong
H
Gillespie
Julia M A
JM
MacMillan
Meeka
M
Belsham
Denise D
DD
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051863:Host Cell Factor C1; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D009478:Neuropeptide Y / Q000235:genetics*; D050811:Octamer Transcription Factor-1; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D037941:Silencer Elements, Transcriptional; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D009478:Neuropeptide Y; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; C083400:PBX1 protein, human