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Huntingtin facilitates polycomb repressive complex 2.

Seong IS, Woda JM, Song JJ, Lloret A, Abeyrathne PD, Woo CJ, Gregory G, Lee JM, Wheeler VC, Walz T, Kingston RE, Gusella JF, Conlon RA, MacDonald ME - Hum. Mol. Genet. (2009)

Bottom Line: Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large alpha-helical domains.Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9.Knowledge of full-length huntingtin's alpha-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Genetic Research, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA.

ABSTRACT
Huntington's disease (HD) is caused by expansion of the polymorphic polyglutamine segment in the huntingtin protein. Full-length huntingtin is thought to be a predominant HEAT repeat alpha-solenoid, implying a role as a facilitator of macromolecular complexes. Here we have investigated huntingtin's domain structure and potential intersection with epigenetic silencer polycomb repressive complex 2 (PRC2), suggested by shared embryonic deficiency phenotypes. Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large alpha-helical domains. Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9. Supporting a direct stimulatory role, full-length recombinant huntingtin significantly increased the histone H3K27 tri-methylase activity of reconstituted PRC2 in vitro, and structure-function analysis demonstrated that the polyglutamine region augmented full-length huntingtin PRC2 stimulation, both in Hdh(Q111) EBs and in vitro, with reconstituted PRC2. Knowledge of full-length huntingtin's alpha-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region.

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Huntingtin associated with PRC2 in the nucleus. (A) Fluorescent microscope images of an immunostained section of a day 4 Hdh+/Hdh+ wild-type (WT) EB, demonstrating the nuclear conformation of huntingtin (Htt), detected by AP194 antibody (8), and anti-Ezh2-signal in DAPI-stained nuclei (merge). (B) Immunoblot showing co-immunoprecipitation of Ezh2 and Suz12 with huntingtin (Htt), from nuclear extracts of day 4 Hdh+/Hdh+ (WT) EBs. (C) Plot of the results of chromatin immunoprecipitation analysis of Hoxb9, demonstrating enrichment of huntingtin (Htt) and trimethylated histone H3K27 (H3K27me3) in day 4 Hdh+/Hdh+ wild-type (WT) EBs, that is not apparent in the absence of huntingtin in Hdhex4/5/Hdhex4/5 (KO) EBs, but is increased in Hdh+/HdhQ111 knock-in (KI) EBs, expressing 111-glutamine huntingtin (n = 3; anti-Htt KO versus WT P < 0.0004; anti-histone H3K27me3 KO versus WT P < 0.0037; anti-Htt KI versus WT P < 0.4267; anti-histone H3K27me3 KI versus WT P < 0.0157).
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DDP524F3: Huntingtin associated with PRC2 in the nucleus. (A) Fluorescent microscope images of an immunostained section of a day 4 Hdh+/Hdh+ wild-type (WT) EB, demonstrating the nuclear conformation of huntingtin (Htt), detected by AP194 antibody (8), and anti-Ezh2-signal in DAPI-stained nuclei (merge). (B) Immunoblot showing co-immunoprecipitation of Ezh2 and Suz12 with huntingtin (Htt), from nuclear extracts of day 4 Hdh+/Hdh+ (WT) EBs. (C) Plot of the results of chromatin immunoprecipitation analysis of Hoxb9, demonstrating enrichment of huntingtin (Htt) and trimethylated histone H3K27 (H3K27me3) in day 4 Hdh+/Hdh+ wild-type (WT) EBs, that is not apparent in the absence of huntingtin in Hdhex4/5/Hdhex4/5 (KO) EBs, but is increased in Hdh+/HdhQ111 knock-in (KI) EBs, expressing 111-glutamine huntingtin (n = 3; anti-Htt KO versus WT P < 0.0004; anti-histone H3K27me3 KO versus WT P < 0.0037; anti-Htt KI versus WT P < 0.4267; anti-histone H3K27me3 KI versus WT P < 0.0157).

Mentions: Developing day 4 EBs were chosen as a system amenable to biochemical analysis, to evaluate the possibility that full-length huntingtin might intersect with PRC2 in the nucleus. Full-length huntingtin was detected by immunoblot analysis in nuclear, as well as cytoplasmic extracts (Supplementary Material, Fig. S3C), and antibody reagent AP194, which immunostained nuclear though not cytoplasmic conformations of full-length huntingtin (8), revealed huntingtin in the nuclei of cells in all three germ-layers, with Ezh2-stain, especially in the outermost endodermal cells (Fig. 3A). Furthermore, the results of analysis of nuclear extracts by gel filtration chromatography demonstrated that full-length huntingtin was co-eluted with PRC2 subunits Ezh2 and Suz12 (Supplementary Material, Fig. S3D). Analysis by co-immunoprecipitation, with specific antibody reagents, yielded a proportion of full-length huntingtin, with Ezh2 and Suz12, as revealed by immunoblot analysis of the precipitated proteins shown in Figure 3B. In addition, as summarized in Figure 3C, chromatin immunoprecipitation (ChIP), with anti-huntingtin or anti-histone H3K27me3, enriched Hoxb9 sequences from wild-type, though not from huntingtin day 4 EB nuclei, thereby placing huntingtin at Hoxb9 chromatin in wild-type cells and supporting a functional role for huntingtin in stimulating histone H3K27 trimethylation.


Huntingtin facilitates polycomb repressive complex 2.

Seong IS, Woda JM, Song JJ, Lloret A, Abeyrathne PD, Woo CJ, Gregory G, Lee JM, Wheeler VC, Walz T, Kingston RE, Gusella JF, Conlon RA, MacDonald ME - Hum. Mol. Genet. (2009)

Huntingtin associated with PRC2 in the nucleus. (A) Fluorescent microscope images of an immunostained section of a day 4 Hdh+/Hdh+ wild-type (WT) EB, demonstrating the nuclear conformation of huntingtin (Htt), detected by AP194 antibody (8), and anti-Ezh2-signal in DAPI-stained nuclei (merge). (B) Immunoblot showing co-immunoprecipitation of Ezh2 and Suz12 with huntingtin (Htt), from nuclear extracts of day 4 Hdh+/Hdh+ (WT) EBs. (C) Plot of the results of chromatin immunoprecipitation analysis of Hoxb9, demonstrating enrichment of huntingtin (Htt) and trimethylated histone H3K27 (H3K27me3) in day 4 Hdh+/Hdh+ wild-type (WT) EBs, that is not apparent in the absence of huntingtin in Hdhex4/5/Hdhex4/5 (KO) EBs, but is increased in Hdh+/HdhQ111 knock-in (KI) EBs, expressing 111-glutamine huntingtin (n = 3; anti-Htt KO versus WT P < 0.0004; anti-histone H3K27me3 KO versus WT P < 0.0037; anti-Htt KI versus WT P < 0.4267; anti-histone H3K27me3 KI versus WT P < 0.0157).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2807366&req=5

DDP524F3: Huntingtin associated with PRC2 in the nucleus. (A) Fluorescent microscope images of an immunostained section of a day 4 Hdh+/Hdh+ wild-type (WT) EB, demonstrating the nuclear conformation of huntingtin (Htt), detected by AP194 antibody (8), and anti-Ezh2-signal in DAPI-stained nuclei (merge). (B) Immunoblot showing co-immunoprecipitation of Ezh2 and Suz12 with huntingtin (Htt), from nuclear extracts of day 4 Hdh+/Hdh+ (WT) EBs. (C) Plot of the results of chromatin immunoprecipitation analysis of Hoxb9, demonstrating enrichment of huntingtin (Htt) and trimethylated histone H3K27 (H3K27me3) in day 4 Hdh+/Hdh+ wild-type (WT) EBs, that is not apparent in the absence of huntingtin in Hdhex4/5/Hdhex4/5 (KO) EBs, but is increased in Hdh+/HdhQ111 knock-in (KI) EBs, expressing 111-glutamine huntingtin (n = 3; anti-Htt KO versus WT P < 0.0004; anti-histone H3K27me3 KO versus WT P < 0.0037; anti-Htt KI versus WT P < 0.4267; anti-histone H3K27me3 KI versus WT P < 0.0157).
Mentions: Developing day 4 EBs were chosen as a system amenable to biochemical analysis, to evaluate the possibility that full-length huntingtin might intersect with PRC2 in the nucleus. Full-length huntingtin was detected by immunoblot analysis in nuclear, as well as cytoplasmic extracts (Supplementary Material, Fig. S3C), and antibody reagent AP194, which immunostained nuclear though not cytoplasmic conformations of full-length huntingtin (8), revealed huntingtin in the nuclei of cells in all three germ-layers, with Ezh2-stain, especially in the outermost endodermal cells (Fig. 3A). Furthermore, the results of analysis of nuclear extracts by gel filtration chromatography demonstrated that full-length huntingtin was co-eluted with PRC2 subunits Ezh2 and Suz12 (Supplementary Material, Fig. S3D). Analysis by co-immunoprecipitation, with specific antibody reagents, yielded a proportion of full-length huntingtin, with Ezh2 and Suz12, as revealed by immunoblot analysis of the precipitated proteins shown in Figure 3B. In addition, as summarized in Figure 3C, chromatin immunoprecipitation (ChIP), with anti-huntingtin or anti-histone H3K27me3, enriched Hoxb9 sequences from wild-type, though not from huntingtin day 4 EB nuclei, thereby placing huntingtin at Hoxb9 chromatin in wild-type cells and supporting a functional role for huntingtin in stimulating histone H3K27 trimethylation.

Bottom Line: Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large alpha-helical domains.Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9.Knowledge of full-length huntingtin's alpha-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region.

View Article: PubMed Central - PubMed

Affiliation: Center for Human Genetic Research, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA.

ABSTRACT
Huntington's disease (HD) is caused by expansion of the polymorphic polyglutamine segment in the huntingtin protein. Full-length huntingtin is thought to be a predominant HEAT repeat alpha-solenoid, implying a role as a facilitator of macromolecular complexes. Here we have investigated huntingtin's domain structure and potential intersection with epigenetic silencer polycomb repressive complex 2 (PRC2), suggested by shared embryonic deficiency phenotypes. Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large alpha-helical domains. Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9. Supporting a direct stimulatory role, full-length recombinant huntingtin significantly increased the histone H3K27 tri-methylase activity of reconstituted PRC2 in vitro, and structure-function analysis demonstrated that the polyglutamine region augmented full-length huntingtin PRC2 stimulation, both in Hdh(Q111) EBs and in vitro, with reconstituted PRC2. Knowledge of full-length huntingtin's alpha-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region.

Show MeSH
Related in: MedlinePlus