Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter.
BACKGROUND
Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of oxidized methionine residues. Most organisms that were genetically modified to lack the MsrA gene have shown shortening of their life span. Methionine sulfoxide reductases B (MsrB) proteins codified by three separate genes, named MsrB1, MsrB2, and MsrB3, are included in the Msrs system. To date, the mechanisms responsible for the transcriptional regulation of MsrB genes have not been reported. The aim of this study was to investigate the regulation of MsrB1 selenoprotein levels through transcriptional regulation of the MsrB1 gene in MDA-MB231 and MCF-7 breast carcinoma cell lines.


RESULTS
A MsrB1 gene promoter is located 169 base pairs upstream from the transcription start site. It contains three Sp1 binding sites which are sufficient for maximal promoter activity in transient transfection experiments. High levels of MsrB1 transcript, protein and promoter activity were detected in low metastatic MCF7 human breast cancer cells. On the contrary, very low levels of both MsrB1 transcript and promoter activity were detected in the highly metastatic counterpart MDA-MB231 cells.A pivotal role for Sp1 in the constitutive expression of the MsrB1 gene was demonstrated through transient expression of mutant MsrB1 promoter-reporter gene constructs and chromatin immunoprecipitation experiments. Since Sp1 is ubiquitously expressed, these sites, while necessary, are not sufficient to explain the patterns of gene expression of MsrB1 in various human breast cancer cells. MDA-MB231 cells can be induced to express MsrB1 by treatment with 5-Aza-2'-deoxycytidine, a demethylating agent. Therefore, the MsrB1 promoter is controlled by epigenetic modifications.


CONCLUSIONS
The results of this study provide the first insights into the transcriptional regulation of the human MsrB1 gene, including the discovery that the Sp1 transcription factor may play a central role in its expression. We also demonstrated that the MsrB1 promoter activity appears to be controlled by epigenetic modifications such as methylation.
De Luca
Antonella
A
Department of Biomedical Sciences, University of Chieti G, D'Annunzio School of Medicine, and Unit of Gene Regulation, Center of Excellence on Aging, G, D'Annunzio University Foundation, Chieti, Italy. atdeluca@unich.it <atdeluca@unich.it>
Sacchetta
Paolo
P
Nieddu
Marzia
M
Di Ilio
Carmine
C
Favaloro
Bartolo
B
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D051150:Methionine Sulfoxide Reductases; D008840:Microfilament Proteins; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D008840:Microfilament Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D010088:Oxidoreductases; C120376:MSRB2 protein, human; D051150:Methionine Sulfoxide Reductases