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Promoters are differentially sensitive to N-terminal mutant huntingtin-mediated transcriptional repression.

Hogel M, Laprairie RB, Denovan-Wright EM - PLoS ONE (2012)

Bottom Line: Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity.We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later.Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neurobiology, Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
Huntington's disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients, animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression. An understanding of the effects of mHtt may lead to strategies to slow the progression of HD. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of the current models using two N-mHtt-affected promoters in two cell models of HD using overexpression of known N-mHtt-interacting transcription factors, promoter deletion and mutation analyses and in vitro promoter binding assays. Consequently, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at promoters. In this model, N-mHtt interacts with multiple partners whose presence and affinity for N-mHtt influence the severity of gene dysregulation. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

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CMV and TK activity were decreased in N548hd cells.CMV-driven firefly luciferase activity (A) and TK-driven Renilla luciferase activity (B) were normalized to total protein in cell lysates. * P<0.05 relative to N548wt as determine by two-tailed t-test. Data are shown as mean ± S.E.M. n = 8 per cell type.
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pone-0041152-g001: CMV and TK activity were decreased in N548hd cells.CMV-driven firefly luciferase activity (A) and TK-driven Renilla luciferase activity (B) were normalized to total protein in cell lysates. * P<0.05 relative to N548wt as determine by two-tailed t-test. Data are shown as mean ± S.E.M. n = 8 per cell type.

Mentions: Reporter plasmids under the control of the CMV and TK promoters were co-transfected into both N548wt and N548hd cells. The luciferase activity driven by the two reporter plasmids was quantified 24 h following transfection using the Dual-Luciferase Reporter (DLR) Assay. Transcription driven by both the CMV and the TK promoters was significantly lower in N548hd cells relative to activity in N548wt cells (Fig. 1). This result suggested that the CMV and TK promoters, when expressed in the N548 cell lines, provide a model for studying the inhibitory effects of N-mHtt on transcription, as well as for comparing and contrasting the effect of N-mHtt on different promoters.


Promoters are differentially sensitive to N-terminal mutant huntingtin-mediated transcriptional repression.

Hogel M, Laprairie RB, Denovan-Wright EM - PLoS ONE (2012)

CMV and TK activity were decreased in N548hd cells.CMV-driven firefly luciferase activity (A) and TK-driven Renilla luciferase activity (B) were normalized to total protein in cell lysates. * P<0.05 relative to N548wt as determine by two-tailed t-test. Data are shown as mean ± S.E.M. n = 8 per cell type.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3399790&req=5

pone-0041152-g001: CMV and TK activity were decreased in N548hd cells.CMV-driven firefly luciferase activity (A) and TK-driven Renilla luciferase activity (B) were normalized to total protein in cell lysates. * P<0.05 relative to N548wt as determine by two-tailed t-test. Data are shown as mean ± S.E.M. n = 8 per cell type.
Mentions: Reporter plasmids under the control of the CMV and TK promoters were co-transfected into both N548wt and N548hd cells. The luciferase activity driven by the two reporter plasmids was quantified 24 h following transfection using the Dual-Luciferase Reporter (DLR) Assay. Transcription driven by both the CMV and the TK promoters was significantly lower in N548hd cells relative to activity in N548wt cells (Fig. 1). This result suggested that the CMV and TK promoters, when expressed in the N548 cell lines, provide a model for studying the inhibitory effects of N-mHtt on transcription, as well as for comparing and contrasting the effect of N-mHtt on different promoters.

Bottom Line: Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity.We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later.Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neurobiology, Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
Huntington's disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients, animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression. An understanding of the effects of mHtt may lead to strategies to slow the progression of HD. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of the current models using two N-mHtt-affected promoters in two cell models of HD using overexpression of known N-mHtt-interacting transcription factors, promoter deletion and mutation analyses and in vitro promoter binding assays. Consequently, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at promoters. In this model, N-mHtt interacts with multiple partners whose presence and affinity for N-mHtt influence the severity of gene dysregulation. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

Show MeSH
Related in: MedlinePlus