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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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β-Synuclein 5′- genomic structure and basal promoter activity.(A) Depicts the 5′-genomic structure of human β-Synuclein gene. Exons are depicted as closed boxes, and 5′-region and introns are black lines. Black box indicates the translational start site in exon 3. Lower panel, luciferase constructs ranging across the 5′-region. * Indicates metal response element (MRE- TGCGCTC). (B & C) Reporter gene assays using Dual-Luciferase™ shows the basal activity of β-Syn promoter fragments and empty pGL3basic vector in SH-SY5Y and U87 MG cells respectively. RLU  =  relative luciferase units.
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pone-0017354-g001: β-Synuclein 5′- genomic structure and basal promoter activity.(A) Depicts the 5′-genomic structure of human β-Synuclein gene. Exons are depicted as closed boxes, and 5′-region and introns are black lines. Black box indicates the translational start site in exon 3. Lower panel, luciferase constructs ranging across the 5′-region. * Indicates metal response element (MRE- TGCGCTC). (B & C) Reporter gene assays using Dual-Luciferase™ shows the basal activity of β-Syn promoter fragments and empty pGL3basic vector in SH-SY5Y and U87 MG cells respectively. RLU  =  relative luciferase units.

Mentions: The putative β-syn promoter region was assessed using a Dual-Luciferase® reporter assay system. The genomic organisation of β-Syn has previously been described [23]. A region spanning ∼10.8 kb 5-prime of the translational start site, pertaining to nine promoter fragments, were analysed (Figure 1A). Parallel reporter gene assays of the nine constructs demonstrated a significant difference in basal transcription rate, in SH-SY5Y cells, between pGL3b −1.1/−0.6 kb and the other β-syn promoter constructs, which had minimal or negligible activity above background (Figure 1B). Background is measured by empty pGL3basic vector. All the β-Syn promoter constructs had minimal or negligible activity in the U-87 MG astrocytoma cells (Figure 1C).


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)

β-Synuclein 5′- genomic structure and basal promoter activity.(A) Depicts the 5′-genomic structure of human β-Synuclein gene. Exons are depicted as closed boxes, and 5′-region and introns are black lines. Black box indicates the translational start site in exon 3. Lower panel, luciferase constructs ranging across the 5′-region. * Indicates metal response element (MRE- TGCGCTC). (B & C) Reporter gene assays using Dual-Luciferase™ shows the basal activity of β-Syn promoter fragments and empty pGL3basic vector in SH-SY5Y and U87 MG cells respectively. RLU  =  relative luciferase units.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017354-g001: β-Synuclein 5′- genomic structure and basal promoter activity.(A) Depicts the 5′-genomic structure of human β-Synuclein gene. Exons are depicted as closed boxes, and 5′-region and introns are black lines. Black box indicates the translational start site in exon 3. Lower panel, luciferase constructs ranging across the 5′-region. * Indicates metal response element (MRE- TGCGCTC). (B & C) Reporter gene assays using Dual-Luciferase™ shows the basal activity of β-Syn promoter fragments and empty pGL3basic vector in SH-SY5Y and U87 MG cells respectively. RLU  =  relative luciferase units.
Mentions: The putative β-syn promoter region was assessed using a Dual-Luciferase® reporter assay system. The genomic organisation of β-Syn has previously been described [23]. A region spanning ∼10.8 kb 5-prime of the translational start site, pertaining to nine promoter fragments, were analysed (Figure 1A). Parallel reporter gene assays of the nine constructs demonstrated a significant difference in basal transcription rate, in SH-SY5Y cells, between pGL3b −1.1/−0.6 kb and the other β-syn promoter constructs, which had minimal or negligible activity above background (Figure 1B). Background is measured by empty pGL3basic vector. All the β-Syn promoter constructs had minimal or negligible activity in the U-87 MG astrocytoma cells (Figure 1C).

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