Mutations in the
alpha-synuclein gene (SNCA) have been implicated in familial Parkinson's
disease (PD) while certain polymorphic alleles at a microsatellite repeat,
NACP-Rep1, located approximately 10 kb upstream of the gene, have been
associated with sporadic PD. In order to study the regulation of the human
alpha-synuclein gene, we performed a deletion analysis of 10.7 kb upstream
of the translational start site, using the luciferase reporter assay in
293T cells and the neuroblastoma cell line SH-SY5Y. The shortest fragment,
400 bp upstream of the transcriptional start site, was sufficient for
transcription in both cell lines. The other constructs led to variable
expression levels, with some showing maximum expression and others showing
nearly complete extinction of expression. An 880 bp fragment located
approximately 10 kb upstream of the gene and containing the NACP-Rep1
polymorphism, was shown to be necessary for normal expression. Additional
analysis of the NACP-Rep1 locus and surrounding DNA suggested that two
domains flanking the repeat interact to enhance expression while the
repeat acts as a negative modulator. Next, we measured the activity of the
entire 10.7 kb upstream region in the luciferase reporter assay when each
of our different NACP-Rep1 alleles were present. The expression levels
varied very significantly among the different alleles over a 3-fold range
in the SH-SY5Y cells but showed little or no significant variation in the
293T cells. Given that even small changes in alpha-synuclein expression
may, over many decades, predispose to PD, the association of different
NACP-Rep1 alleles with PD may be a consequence of polymorphic differences
in transcriptional regulation of alpha-synuclein expression resulting from
different NACP-Rep1 alleles.
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Mutations in the
alpha-synuclein gene (SNCA) have been implicated in familial Parkinson's
disease (PD) while certain polymorphic alleles at a microsatellite repeat,
NACP-Rep1, located approximately 10 kb upstream of the gene, have been
associated with sporadic PD. In order to study the regulation of the human
alpha-synuclein gene, we performed a deletion analysis of 10.7 kb upstream
of the translational start site, using the luciferase reporter assay in
293T cells and the neuroblastoma cell line SH-SY5Y. The shortest fragment,
400 bp upstream of the transcriptional start site, was sufficient for
transcription in both cell lines. The other constructs led to variable
expression levels, with some showing maximum expression and others showing
nearly complete extinction of expression. An 880 bp fragment located
approximately 10 kb upstream of the gene and containing the NACP-Rep1
polymorphism, was shown to be necessary for normal expression. Additional
analysis of the NACP-Rep1 locus and surrounding DNA suggested that two
domains flanking the repeat interact to enhance expression while the
repeat acts as a negative modulator. Next, we measured the activity of the
entire 10.7 kb upstream region in the luciferase reporter assay when each
of our different NACP-Rep1 alleles were present. The expression levels
varied very significantly among the different alleles over a 3-fold range
in the SH-SY5Y cells but showed little or no significant variation in the
293T cells. Given that even small changes in alpha-synuclein expression
may, over many decades, predispose to PD, the association of different
NACP-Rep1 alleles with PD may be a consequence of polymorphic differences
in transcriptional regulation of alpha-synuclein expression resulting from
different NACP-Rep1 alleles.
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