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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-Syn promoter regulation.The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in cells stably transfected with pcDNA3.1+ or pcDNA3.1+MTF-1 in (A) SH-SY5Y and (B) U87 MG cells. (C) The activity of β-Syn promoter fragments pGL3b −3.7 kb/ATG, −0.9 kb/ATG and −6.0/−0.9 kb in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (D) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb with mutation inserted at −774 bp in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (E) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in the presence of 0 µM, 10 µM & 50 µM copper in cells stably transfected with either pcDNA3.1+ or pcDNA3.1+MTF-1 The pGL3basic background activity is shown in graphs where the promoter activity is low. *  =  p<0.05, all the rest were non-significant. RLU  =  relative luciferase units. Light grey bars  =  pGL3basic; dark grey bars  =  pcDNA3.1+; black bars  =  pcDNA3.1+MTF-1.
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pone-0017354-g002: β-Syn promoter regulation.The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in cells stably transfected with pcDNA3.1+ or pcDNA3.1+MTF-1 in (A) SH-SY5Y and (B) U87 MG cells. (C) The activity of β-Syn promoter fragments pGL3b −3.7 kb/ATG, −0.9 kb/ATG and −6.0/−0.9 kb in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (D) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb with mutation inserted at −774 bp in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (E) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in the presence of 0 µM, 10 µM & 50 µM copper in cells stably transfected with either pcDNA3.1+ or pcDNA3.1+MTF-1 The pGL3basic background activity is shown in graphs where the promoter activity is low. *  =  p<0.05, all the rest were non-significant. RLU  =  relative luciferase units. Light grey bars  =  pGL3basic; dark grey bars  =  pcDNA3.1+; black bars  =  pcDNA3.1+MTF-1.

Mentions: To investigate the regulation of β-Syn −1.1/−0.6 kb, we performed a transcriptional binding site analysis using the Transcription Element Search System (TESS) [24] and identified a putative metal transcription factor-1 (MTF-1) binding site pertaining to a metal response element (MRE-TGCGCTC). To explore this further we looked at the effect of cells over-expressing MTF-1 on the β-Syn −1.1/−0.6 kb promoter fragment. Figure 2A shows that in cells over-expressing pcDNA3.1+MTF-1 there was a statistically significant increase in β-Syn −1.1/−0.6 kb promoter stably-transfected with the empty expression vector, pcDNA3.1+. We also tested the affect of MTF-1 on −1.1/−0.6 kb in U-87 MG cells (Figure 2B) and although the basal activity of −1.1/−0.6 kb was only slightly above empty vector (pGL3basic), there was a similar trend for that observed in the SH-SY5Y cells. We also investigated whether MTF-1 affected two larger promoter constructs containing the MRE (−3.7kb/ATG & -0.9 kb/ATG) and a larger fragment without the MRE (−6.0/−0.9 kb) (Figure 2C). All three constructs were not significantly affected by MTF-1 over-expression, however, the two MRE-containing promoter fragments did trend to the effect observed for the β-Syn −1.1/−0.6 kb construct. This less dramatic effect on the two larger MRE-containing promoter fragments could be due to the presence of repressor elements counteracting the effect of MTF-1 on the MRE, which may also explain the initial lower basal activity observed for these two promoter constructs (Figure 1B).


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-Syn promoter regulation.The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in cells stably transfected with pcDNA3.1+ or pcDNA3.1+MTF-1 in (A) SH-SY5Y and (B) U87 MG cells. (C) The activity of β-Syn promoter fragments pGL3b −3.7 kb/ATG, −0.9 kb/ATG and −6.0/−0.9 kb in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (D) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb with mutation inserted at −774 bp in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (E) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in the presence of 0 µM, 10 µM & 50 µM copper in cells stably transfected with either pcDNA3.1+ or pcDNA3.1+MTF-1 The pGL3basic background activity is shown in graphs where the promoter activity is low. *  =  p<0.05, all the rest were non-significant. RLU  =  relative luciferase units. Light grey bars  =  pGL3basic; dark grey bars  =  pcDNA3.1+; black bars  =  pcDNA3.1+MTF-1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046239&req=5

pone-0017354-g002: β-Syn promoter regulation.The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in cells stably transfected with pcDNA3.1+ or pcDNA3.1+MTF-1 in (A) SH-SY5Y and (B) U87 MG cells. (C) The activity of β-Syn promoter fragments pGL3b −3.7 kb/ATG, −0.9 kb/ATG and −6.0/−0.9 kb in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (D) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb with mutation inserted at −774 bp in SH-SY5Y cells over-expressing pcDNA3.1+ or pcDNA3.1+MTF-1. (E) The activity of β-Syn promoter fragment pGL3b −1.1/−0.6 kb in the presence of 0 µM, 10 µM & 50 µM copper in cells stably transfected with either pcDNA3.1+ or pcDNA3.1+MTF-1 The pGL3basic background activity is shown in graphs where the promoter activity is low. *  =  p<0.05, all the rest were non-significant. RLU  =  relative luciferase units. Light grey bars  =  pGL3basic; dark grey bars  =  pcDNA3.1+; black bars  =  pcDNA3.1+MTF-1.
Mentions: To investigate the regulation of β-Syn −1.1/−0.6 kb, we performed a transcriptional binding site analysis using the Transcription Element Search System (TESS) [24] and identified a putative metal transcription factor-1 (MTF-1) binding site pertaining to a metal response element (MRE-TGCGCTC). To explore this further we looked at the effect of cells over-expressing MTF-1 on the β-Syn −1.1/−0.6 kb promoter fragment. Figure 2A shows that in cells over-expressing pcDNA3.1+MTF-1 there was a statistically significant increase in β-Syn −1.1/−0.6 kb promoter stably-transfected with the empty expression vector, pcDNA3.1+. We also tested the affect of MTF-1 on −1.1/−0.6 kb in U-87 MG cells (Figure 2B) and although the basal activity of −1.1/−0.6 kb was only slightly above empty vector (pGL3basic), there was a similar trend for that observed in the SH-SY5Y cells. We also investigated whether MTF-1 affected two larger promoter constructs containing the MRE (−3.7kb/ATG & -0.9 kb/ATG) and a larger fragment without the MRE (−6.0/−0.9 kb) (Figure 2C). All three constructs were not significantly affected by MTF-1 over-expression, however, the two MRE-containing promoter fragments did trend to the effect observed for the β-Syn −1.1/−0.6 kb construct. This less dramatic effect on the two larger MRE-containing promoter fragments could be due to the presence of repressor elements counteracting the effect of MTF-1 on the MRE, which may also explain the initial lower basal activity observed for these two promoter constructs (Figure 1B).

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