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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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Transcription driven by the−99 CMV promoter was inhibited by N-mHtt. The −772 CMV promoter was sequentially deleted to 297 (−297 CMV) and 99 (−99 CMV) bp upstream of the transcription start site and promoters were inserted into the pGL3-Basic reporter plasmid. Activity of these plasmids in N548wt and N548hd cells is shown normalized to total protein. * P<0.05 relative to −772 CMV. # P<0.05 relative to −772 CMV and −297 CMV. ∧ P<0.05 relative to −772 CMV, −297 CMV and −99 CMV ∼ P<0.05 relative to N548wt cells as determined by two-way ANOVA followed by a Tamhane’s T2 post-hoc test for unequal variance to analyze effect of promoter deletion, and one-tailed t-test to analyze cell-type effect. Data are shown as mean ± S.E.M. n = 8 per data set.
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pone-0041152-g002: Transcription driven by the−99 CMV promoter was inhibited by N-mHtt. The −772 CMV promoter was sequentially deleted to 297 (−297 CMV) and 99 (−99 CMV) bp upstream of the transcription start site and promoters were inserted into the pGL3-Basic reporter plasmid. Activity of these plasmids in N548wt and N548hd cells is shown normalized to total protein. * P<0.05 relative to −772 CMV. # P<0.05 relative to −772 CMV and −297 CMV. ∧ P<0.05 relative to −772 CMV, −297 CMV and −99 CMV ∼ P<0.05 relative to N548wt cells as determined by two-way ANOVA followed by a Tamhane’s T2 post-hoc test for unequal variance to analyze effect of promoter deletion, and one-tailed t-test to analyze cell-type effect. Data are shown as mean ± S.E.M. n = 8 per data set.

Mentions: To determine if the region of the CMV promoter required for transcriptional repression in the presence of N-mHtt could be identified, fragments of the CMV promoter spanning 772, 297 and 99 bp 5′ to the transcription start site (−772 CMV, −297 CMV, and −99 CMV) were transfected into N548wt and N548hd cells. The sequential deletion of the CMV promoter resulted in progressively lower luciferase activity (Fig. 2), indicating that activator elements reside in the region between 100 and 772 bp on the CMV promoter. Activity of the −99 CMV promoter was significantly higher than the background activity produced by empty pGL3-Basic plasmid, which indicated that deletion of the CMV promoter to the 99 bp proximal to the transcription start site did not completely eliminate transcriptional activity. With regards to the inhibitory effects of N-mHtt on transcription driven by the CMV promoter, luciferase activity was significantly lower in N548hd cells than in N548wt cells for all of the CMV promoter deletion fragments tested. There was no difference in the activity of the pGL3-Basic control plasmid in N548hd cells compared to N548wt cells, suggesting that N-mHtt-mediated repression of luciferase activity was specific to the CMV promoter. As the sequential reduction of the CMV promoter did not alleviate N-mHtt-mediated transcriptional dysregulation, it appeared that N-mHtt-mediated repression of the CMV promoter was localized to the region within 99 bp of the transcription start site.


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

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

Transcription driven by the−99 CMV promoter was inhibited by N-mHtt. The −772 CMV promoter was sequentially deleted to 297 (−297 CMV) and 99 (−99 CMV) bp upstream of the transcription start site and promoters were inserted into the pGL3-Basic reporter plasmid. Activity of these plasmids in N548wt and N548hd cells is shown normalized to total protein. * P<0.05 relative to −772 CMV. # P<0.05 relative to −772 CMV and −297 CMV. ∧ P<0.05 relative to −772 CMV, −297 CMV and −99 CMV ∼ P<0.05 relative to N548wt cells as determined by two-way ANOVA followed by a Tamhane’s T2 post-hoc test for unequal variance to analyze effect of promoter deletion, and one-tailed t-test to analyze cell-type effect. Data are shown as mean ± S.E.M. n = 8 per data set.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0041152-g002: Transcription driven by the−99 CMV promoter was inhibited by N-mHtt. The −772 CMV promoter was sequentially deleted to 297 (−297 CMV) and 99 (−99 CMV) bp upstream of the transcription start site and promoters were inserted into the pGL3-Basic reporter plasmid. Activity of these plasmids in N548wt and N548hd cells is shown normalized to total protein. * P<0.05 relative to −772 CMV. # P<0.05 relative to −772 CMV and −297 CMV. ∧ P<0.05 relative to −772 CMV, −297 CMV and −99 CMV ∼ P<0.05 relative to N548wt cells as determined by two-way ANOVA followed by a Tamhane’s T2 post-hoc test for unequal variance to analyze effect of promoter deletion, and one-tailed t-test to analyze cell-type effect. Data are shown as mean ± S.E.M. n = 8 per data set.
Mentions: To determine if the region of the CMV promoter required for transcriptional repression in the presence of N-mHtt could be identified, fragments of the CMV promoter spanning 772, 297 and 99 bp 5′ to the transcription start site (−772 CMV, −297 CMV, and −99 CMV) were transfected into N548wt and N548hd cells. The sequential deletion of the CMV promoter resulted in progressively lower luciferase activity (Fig. 2), indicating that activator elements reside in the region between 100 and 772 bp on the CMV promoter. Activity of the −99 CMV promoter was significantly higher than the background activity produced by empty pGL3-Basic plasmid, which indicated that deletion of the CMV promoter to the 99 bp proximal to the transcription start site did not completely eliminate transcriptional activity. With regards to the inhibitory effects of N-mHtt on transcription driven by the CMV promoter, luciferase activity was significantly lower in N548hd cells than in N548wt cells for all of the CMV promoter deletion fragments tested. There was no difference in the activity of the pGL3-Basic control plasmid in N548hd cells compared to N548wt cells, suggesting that N-mHtt-mediated repression of luciferase activity was specific to the CMV promoter. As the sequential reduction of the CMV promoter did not alleviate N-mHtt-mediated transcriptional dysregulation, it appeared that N-mHtt-mediated repression of the CMV promoter was localized to the region within 99 bp of the transcription start site.

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