Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells.
(O)estrogen receptor-alpha (ERalpha), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERalpha gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at -117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of gamma-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PF a and PF b, that do not present binding sites for known transcription factors and that are involved in a strong DNA-protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERalpha gene expression in bone. These data provide evidence for different promoter usage of the ERalpha gene through the recruitment of tissue-specific transcription activators and co-regulators.
Lambertini
Elisabetta
E
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Ferrara, Via L. Borsari 46, Italy.
Penolazzi
Letizia
L
Giordano
Silvia
S
Del Senno
Laura
L
Piva
Roberta
R
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics; D047628:Estrogen Receptor alpha; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases