Analysis of a repressor region in the human neuropeptide Y gene that binds Oct-1 and Pbx-1 in GT1-7 neurons

doi:10.1016/S0006-291X(03)01289-0

Biochemical and Biophysical Research Communications

Volume 307, Issue 4, 8 August 2003, Pages 847-854

https://doi.org/10.1016/S0006-291X(03)01289-0 Get rights and content

Abstract

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 −922 bp 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.

Section snippets

Materials and methods

Cell culture and reagents. GT1-7 and baby hamster kidney (BHK) cells were maintained in monolayer in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Burlington, Ont.) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 4.5 mg/ml glucose, and penicillin/streptomycin and maintained at 37 °C in an atmosphere of 5% CO₂ as previously described [18]. Luciferin was obtained from ICN Biomedicals (Montreal, PQ). All primers and oligonucleotides were synthesized by ACGT

A potential repressor region is located between −1078 and −867 of the human NPY gene 5^′ regulatory region and binds GT1-7 nuclear proteins

The endogenous NPY gene is not expressed at detectable levels in the GT1-7 hypothalamic neurons. To study the role of transcription as a regulatory mechanism involved in the repression of NPY gene expression in the GT1-7 cells, a series of reporter plasmids representing sequential deletions of the human −1078 NPY 5^′ regulatory region were generated and transiently transfected into GT1-7 cells. Interestingly, many of the regions were active in the GT1-7 neurons, as expression reached up to

Discussion

The transcriptional control of neuronal gene expression is not well understood, but knowledge of the mechanisms involved in this process holds much promise for therapeutic strategies. While the transcriptional mechanisms required for activation of gene expression have received attention, the repression of gene activity and the transcription factors involved in this process are not well known. Previous studies have looked at cis-acting elements required for NPY gene expression and characterized

Acknowledgements

We thank Dr. Pamela L. Mellon, University of California, San Diego, for generously providing the GT1-7 cells. Thanks to Dr. Bernardo Yusta for critical reading of the manuscript. This work was supported by the Canadian Institutes for Health Research (CIHR). D.D.B. is a CIHR Scholar and a Canada Foundation for Innovation Researcher. H.C. was supported by a Premier’s Research Excellence Award to D.D.B.

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Analysis of a repressor region in the human neuropeptide Y gene that binds Oct-1 and Pbx-1 in GT1-7 neurons

Abstract

Section snippets

Materials and methods

A potential repressor region is located between −1078 and −867 of the human NPY gene 5′ regulatory region and binds GT1-7 nuclear proteins

Discussion

Acknowledgements

J. Biol. Chem.

Regul. Pept.

Neurosci. Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

Brain Res. Mol. Brain Res.

Neuron

Cell

Gene Anal. Tech.

Prog. Nucleic Acid Res. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Nature

Endocrinology

A potential repressor region is located between −1078 and −867 of the human NPY gene 5^′ regulatory region and binds GT1-7 nuclear proteins