Biochemical and Biophysical Research Communications
Negative regulation of human parathyroid hormone gene promoter by vitamin D3 through nuclear factor Y☆
Section snippets
Materials and methods
Constructs. The single-stranded 27-deoxynucleotide-long PTH-VDRE (27-VDRE, Fig. 1) [3] and the NF-Y consensus oligodeoxynucleotide (Ycons, Fig. 1) [5], [6], [7], [8] were synthesized with MluI or HindIII overhangs, annealed, and cloned into the respective site of the luciferase plasmid pGL3 (Promega, Madison, USA). The extended 44-deoxynucleotide-long single-stranded sequences (44-VDRE, Fig. 1) were annealed and blunt-end ligated into the SmaI site of the same plasmid. The pGL3 construct
Characterization of protein–DNA interactions of the PTH-VDRE region
To characterize protein–DNA interactions at the PTH-VDRE sequences, we performed EMSA with the different PTH-VDRE probes. The 27-VDRE previously reported to bind VDR [2], [3] formed several protein–DNA complexes with nuclear proteins from GH4C1 cells (Fig. 2). From these complexes, the most slowly migrating complex (arrowhead) was recognized by the VDR N-terminal antibody, whereas none of the complexes was recognized by the Sp1 (Fig. 2A) or NF-Y (Fig. 2B) antibodies. Binding reactions with the
Discussion
Nuclear factor Y (NF-Y) is an abundant transcription factor, which regulates expression of about 30% of all eukaryotic genes. It has a very high requirement for the sequence of CCAAT and its flanking nucleotides for efficient DNA binding. Only nine sequences out of the 178 sequences studied had one nucleotide difference in the CCAAT core and, for these sequences, the binding affinity of NF-Y was lower than that for the intact sequence [7]. In this study, we found that NF-Y bound to the extended
Acknowledgments
We thank Dr. Roberto Mantovani for the NF-YAmut construct, Dr. Henry Kronenberg for the pc301 plasmid, and Dr. Lise Binderup for 1,25(OH)2D3. We also thank Mrs. Maija Hiltunen, Mrs. Hanna Eskelinen, and Miss Liisa Röntynen for skilful technical assistance. This work was supported in part by the grants from the Research and Science Foundation of Farmos Co. (T.J.) and from the Academy of Finland (Grant No. 30566, P.M.).
References (14)
- et al.
5′-Flanking region of the parathyroid hormone gene mediates negative regulation by 1,25-(OH)2 vitamin D3
J. Biol. Chem.
(1988) - et al.
Identification of a transcription factor that binds to the promoter region of the human parathyroid hormone gene
Arch. Biochem. Biophys.
(1999) Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences
J. Mol. Biol.
(1990)- et al.
DNA binding specificity of the CCAAT-binding factor CBF/NF-Y
J. Biol. Chem.
(1997) The molecular biology of the CCAAT-binding factor NF-Y
Gene
(1999)- et al.
Dominant negative analogs of NF-YA
J. Biol. Chem.
(1994) - et al.
Transcriptional activity of the SHP-1 gene in MCF7 cells is differentially regulated by binding of NF-Y factor to two distinct CCAAT-elements
Gene
(2001)
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Involvement of GCMB in the transcriptional regulation of the human parathyroid hormone gene in a parathyroid-derived cell line PT-r. Effects of calcium and 1,25(OH)<inf>2</inf>D<inf>3</inf>
2010, BoneCitation Excerpt :Within this region we found putative binding sites for Pbx1, GATA3, Sp1, Mafs, and NF-Y (Fig. 3A), and one or some of these transcription factors may be working to maintain the basal transcriptional activity of PTH gene [15–18,33–35]. Besides the tissue-specific regulation, PTH gene also receives short-term and dynamic transcriptional regulation by extracellular calcium and 1,25(OH)2D [5,7–12,14,16,20,21,45,46]. Among the factors, plasma calcium is known to exert a strict negative control on PTH secretion, but the effect on PTH gene expression is much less clear.
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Abbreviations: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; CRBP-RXRE, retinoic acid responsive element of the cellular retinol-binding protein II gene; EMSA, electrophoretic mobility shift assay; NF-Y, nuclear factor Y; PTH, parathyroid hormone; RXR, retinoid X receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.
- 1
These authors should be considered as equal first authors.
- 2
Present address: Department of Medical Biochemistry, University of Kuopio, FIN-70211 Kuopio, Finland.
- 3
Present address: University Hospital of Oulu, FIN-90220 Oulu, Finland.