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Transcriptional regulation of the beta-synuclein 5'-promoter metal response element by metal transcription factor-1.

McHugh PC, Wright JA, Brown DR - PLoS ONE (2011)

Bottom Line: This effect of MTF-1 on expression was found to be specific to beta-synuclein when compared to alpha-synuclein.Understanding the regulation of synucleins and how they interact may point to molecular targets that could be manipulated for therapeutic benefit.In this study we showed that MTF-1 differentially controls the expression of beta-synuclein when compared to its homolog alpha-synuclein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom.

ABSTRACT
The progression of many human neurodegenerative disorders is associated with an accumulation of alpha-synuclein. Alpha-synuclein belongs to the homologous synuclein family, which includes beta-synuclein. It has been proposed that beta-synuclein may be a natural regulator of alpha-synuclein. Therefore controlling beta-synuclein expression may control the accumulation of alpha-synuclein and ultimately prevent disease progression. The regulation of synucleins is poorly understood. We investigated the transcriptional regulation of beta-synuclein, with the aim of identifying molecules that differentially control beta-synuclein expression levels. To investigate transcriptional regulation of beta-synuclein, we used reporter gene assays and bioinformatics. We identified a region -1.1/-0.6 kb upstream of the beta-synuclein translational start site to be a key regulatory region of beta-synuclein 5'-promoter activity in human dopaminergic cells (SH-SY5Y). Within this key promoter region we identified a metal response element pertaining to a putative Metal Transcription Factor-1 (MTF-1) binding site. We demonstrated that MTF-1 binds to this 5'-promoter region using EMSA analysis. Moreover, we showed that MTF-1 differentially regulates beta-synuclein promoter binding site, as well as beta-synuclein mRNA and protein expression. This effect of MTF-1 on expression was found to be specific to beta-synuclein when compared to alpha-synuclein. Understanding the regulation of synucleins and how they interact may point to molecular targets that could be manipulated for therapeutic benefit. In this study we showed that MTF-1 differentially controls the expression of beta-synuclein when compared to its homolog alpha-synuclein. This could potentially provide a novel targets or pathways for therapeutic intervention and/or treatment of synucleinopathies.

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EMSA analysis.Nuclear extracts from SH-SY5Y cells were stably transfected with (A & C) pcDNA3.1+MTF-1 or (B) pcDNA3.1+. A & B are probed with normal probe and C with mutant probe. Lane 1 is the migration of free-probe in the absence of nuclear extract and therefore no shift observed. Lane 2 is either the MRE-containing double-stranded DNA probe (A & C) and shows a signal shift due to transcription factor binding or the mutant MRE-containing probe (B) and shows a diminished signal shift. Lane 3 shows that the signal shift can be inhibited from excess non-labelled probe.
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pone-0017354-g003: EMSA analysis.Nuclear extracts from SH-SY5Y cells were stably transfected with (A & C) pcDNA3.1+MTF-1 or (B) pcDNA3.1+. A & B are probed with normal probe and C with mutant probe. Lane 1 is the migration of free-probe in the absence of nuclear extract and therefore no shift observed. Lane 2 is either the MRE-containing double-stranded DNA probe (A & C) and shows a signal shift due to transcription factor binding or the mutant MRE-containing probe (B) and shows a diminished signal shift. Lane 3 shows that the signal shift can be inhibited from excess non-labelled probe.

Mentions: To determine whether the observed MTF-1 regulation of the β-Syn promoter was a consequence of MTF-1 binding to the β-syn promoter MRE or an indirect effect of MTF-1 on the β-syn promoter activity, we performed EMSAs. EMSA analysis showed that in cells over-expressing the transcription factor MTF-1 there was an increase in binding for the normal MRE-containing probe compared to the mutant probe as indicated by the band intensity shift when compared to unlabelled probe (Figure 3A & B). In comparison to pcDNA3.1+ over-expressing cells, this effect was less intense (Figure 3C), although more intense than the mutant probe in cells over-expressing MTF-1, suggesting that endogenous levels of MTF-1 are present. These results show that MTF-1 binds to MRE located in the −1.1/−0.6 kb region of the β-Syn 5′-promoter.


Transcriptional regulation of the beta-synuclein 5'-promoter metal response element by metal transcription factor-1.

McHugh PC, Wright JA, Brown DR - PLoS ONE (2011)

EMSA analysis.Nuclear extracts from SH-SY5Y cells were stably transfected with (A & C) pcDNA3.1+MTF-1 or (B) pcDNA3.1+. A & B are probed with normal probe and C with mutant probe. Lane 1 is the migration of free-probe in the absence of nuclear extract and therefore no shift observed. Lane 2 is either the MRE-containing double-stranded DNA probe (A & C) and shows a signal shift due to transcription factor binding or the mutant MRE-containing probe (B) and shows a diminished signal shift. Lane 3 shows that the signal shift can be inhibited from excess non-labelled probe.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046239&req=5

pone-0017354-g003: EMSA analysis.Nuclear extracts from SH-SY5Y cells were stably transfected with (A & C) pcDNA3.1+MTF-1 or (B) pcDNA3.1+. A & B are probed with normal probe and C with mutant probe. Lane 1 is the migration of free-probe in the absence of nuclear extract and therefore no shift observed. Lane 2 is either the MRE-containing double-stranded DNA probe (A & C) and shows a signal shift due to transcription factor binding or the mutant MRE-containing probe (B) and shows a diminished signal shift. Lane 3 shows that the signal shift can be inhibited from excess non-labelled probe.
Mentions: To determine whether the observed MTF-1 regulation of the β-Syn promoter was a consequence of MTF-1 binding to the β-syn promoter MRE or an indirect effect of MTF-1 on the β-syn promoter activity, we performed EMSAs. EMSA analysis showed that in cells over-expressing the transcription factor MTF-1 there was an increase in binding for the normal MRE-containing probe compared to the mutant probe as indicated by the band intensity shift when compared to unlabelled probe (Figure 3A & B). In comparison to pcDNA3.1+ over-expressing cells, this effect was less intense (Figure 3C), although more intense than the mutant probe in cells over-expressing MTF-1, suggesting that endogenous levels of MTF-1 are present. These results show that MTF-1 binds to MRE located in the −1.1/−0.6 kb region of the β-Syn 5′-promoter.

Bottom Line: This effect of MTF-1 on expression was found to be specific to beta-synuclein when compared to alpha-synuclein.Understanding the regulation of synucleins and how they interact may point to molecular targets that could be manipulated for therapeutic benefit.In this study we showed that MTF-1 differentially controls the expression of beta-synuclein when compared to its homolog alpha-synuclein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom.

ABSTRACT
The progression of many human neurodegenerative disorders is associated with an accumulation of alpha-synuclein. Alpha-synuclein belongs to the homologous synuclein family, which includes beta-synuclein. It has been proposed that beta-synuclein may be a natural regulator of alpha-synuclein. Therefore controlling beta-synuclein expression may control the accumulation of alpha-synuclein and ultimately prevent disease progression. The regulation of synucleins is poorly understood. We investigated the transcriptional regulation of beta-synuclein, with the aim of identifying molecules that differentially control beta-synuclein expression levels. To investigate transcriptional regulation of beta-synuclein, we used reporter gene assays and bioinformatics. We identified a region -1.1/-0.6 kb upstream of the beta-synuclein translational start site to be a key regulatory region of beta-synuclein 5'-promoter activity in human dopaminergic cells (SH-SY5Y). Within this key promoter region we identified a metal response element pertaining to a putative Metal Transcription Factor-1 (MTF-1) binding site. We demonstrated that MTF-1 binds to this 5'-promoter region using EMSA analysis. Moreover, we showed that MTF-1 differentially regulates beta-synuclein promoter binding site, as well as beta-synuclein mRNA and protein expression. This effect of MTF-1 on expression was found to be specific to beta-synuclein when compared to alpha-synuclein. Understanding the regulation of synucleins and how they interact may point to molecular targets that could be manipulated for therapeutic benefit. In this study we showed that MTF-1 differentially controls the expression of beta-synuclein when compared to its homolog alpha-synuclein. This could potentially provide a novel targets or pathways for therapeutic intervention and/or treatment of synucleinopathies.

Show MeSH
Related in: MedlinePlus