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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 stimulated recombinant PRC2 in vitro. (A) Immunoblot showing co-immunoprecipitation of FLAG-huntingtin with recombinant Ezh2 and Suz12 members of reconstituted PRC2 to which huntingtin (Htt) was added (+) or was not added (−). (B) Autoradiogram of bands of 3H-methyl histone H3 produced by reconstituted PRC2, and below the plot of the quantified band intensities, demonstrating that addition of 40 nm recombinant Q23 huntingtin (Htt), but not bovine serum albumin (BSA), or tag-peptide (Tag peptide), significantly stimulated PRC2 activity. (n = 3; *P < 0.05). (C) Autoradiogram showing bands of 3H-methyl histone H3 produced by reconstituted PRC2 in the absence (−) and presence of 2 nm recombinant huntingtins, with different polyglutamine sizes, and below a plot of quantified band intensities, relative to baseline PRC2 activity, demonstrating a progressive increase in huntingtin's stimulation of PRC2 as polyglutamine size is increased (n = 3; Q43Htt or Q32 versus no Htt *P < 0.017; Q43Htt versus Q23Htt **P < 0.045).
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DDP524F4: Huntingtin stimulated recombinant PRC2 in vitro. (A) Immunoblot showing co-immunoprecipitation of FLAG-huntingtin with recombinant Ezh2 and Suz12 members of reconstituted PRC2 to which huntingtin (Htt) was added (+) or was not added (−). (B) Autoradiogram of bands of 3H-methyl histone H3 produced by reconstituted PRC2, and below the plot of the quantified band intensities, demonstrating that addition of 40 nm recombinant Q23 huntingtin (Htt), but not bovine serum albumin (BSA), or tag-peptide (Tag peptide), significantly stimulated PRC2 activity. (n = 3; *P < 0.05). (C) Autoradiogram showing bands of 3H-methyl histone H3 produced by reconstituted PRC2 in the absence (−) and presence of 2 nm recombinant huntingtins, with different polyglutamine sizes, and below a plot of quantified band intensities, relative to baseline PRC2 activity, demonstrating a progressive increase in huntingtin's stimulation of PRC2 as polyglutamine size is increased (n = 3; Q43Htt or Q32 versus no Htt *P < 0.017; Q43Htt versus Q23Htt **P < 0.045).

Mentions: This interpretation was confirmed by the results of in vitro experiments, to determine whether recombinant full-length human huntingtin would interact with and alter the histone H3K27 methyltransferase activity of reconstituted PRC2 in a previously reported in vitro assay (30–33) (see Materials and Methods). As shown in the immunoblot in Figure 4A, full-length FLAG-tag Q23 huntingtin added to recombinant PRC2 was co-immunoprecipitated with Ezh2 and Suz12, and, as illustrated in Figure 4B, the recombinant protein significantly increased PRC2-specific histone H3K27 methylation, as judged by the intensity of bands of incorporated tritium. The stimulatory effect of full-length huntingtin, compared with reactions without huntingtin or with control peptides, was observed over a range of huntingtin (Supplementary Material, Fig. S4A) and nucleosomal array (Supplementary Material, Fig. S4B) concentrations. Furthermore, consistent with the finding that huntingtin was needed to stimulate tri- but not di-methylation of histone H3K27 in vivo (Fig. 2B, Supplementary Material, Fig. S3B), the results of immunoblot analysis of the in vitro PRC2 reaction products demonstrated that recombinant huntingtin specifically enhanced histone H3K27 tri-methylation but not di-methylation (Supplementary Material, Fig. S4C).


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 stimulated recombinant PRC2 in vitro. (A) Immunoblot showing co-immunoprecipitation of FLAG-huntingtin with recombinant Ezh2 and Suz12 members of reconstituted PRC2 to which huntingtin (Htt) was added (+) or was not added (−). (B) Autoradiogram of bands of 3H-methyl histone H3 produced by reconstituted PRC2, and below the plot of the quantified band intensities, demonstrating that addition of 40 nm recombinant Q23 huntingtin (Htt), but not bovine serum albumin (BSA), or tag-peptide (Tag peptide), significantly stimulated PRC2 activity. (n = 3; *P < 0.05). (C) Autoradiogram showing bands of 3H-methyl histone H3 produced by reconstituted PRC2 in the absence (−) and presence of 2 nm recombinant huntingtins, with different polyglutamine sizes, and below a plot of quantified band intensities, relative to baseline PRC2 activity, demonstrating a progressive increase in huntingtin's stimulation of PRC2 as polyglutamine size is increased (n = 3; Q43Htt or Q32 versus no Htt *P < 0.017; Q43Htt versus Q23Htt **P < 0.045).
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Related In: Results  -  Collection

License 1 - License 2
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DDP524F4: Huntingtin stimulated recombinant PRC2 in vitro. (A) Immunoblot showing co-immunoprecipitation of FLAG-huntingtin with recombinant Ezh2 and Suz12 members of reconstituted PRC2 to which huntingtin (Htt) was added (+) or was not added (−). (B) Autoradiogram of bands of 3H-methyl histone H3 produced by reconstituted PRC2, and below the plot of the quantified band intensities, demonstrating that addition of 40 nm recombinant Q23 huntingtin (Htt), but not bovine serum albumin (BSA), or tag-peptide (Tag peptide), significantly stimulated PRC2 activity. (n = 3; *P < 0.05). (C) Autoradiogram showing bands of 3H-methyl histone H3 produced by reconstituted PRC2 in the absence (−) and presence of 2 nm recombinant huntingtins, with different polyglutamine sizes, and below a plot of quantified band intensities, relative to baseline PRC2 activity, demonstrating a progressive increase in huntingtin's stimulation of PRC2 as polyglutamine size is increased (n = 3; Q43Htt or Q32 versus no Htt *P < 0.017; Q43Htt versus Q23Htt **P < 0.045).
Mentions: This interpretation was confirmed by the results of in vitro experiments, to determine whether recombinant full-length human huntingtin would interact with and alter the histone H3K27 methyltransferase activity of reconstituted PRC2 in a previously reported in vitro assay (30–33) (see Materials and Methods). As shown in the immunoblot in Figure 4A, full-length FLAG-tag Q23 huntingtin added to recombinant PRC2 was co-immunoprecipitated with Ezh2 and Suz12, and, as illustrated in Figure 4B, the recombinant protein significantly increased PRC2-specific histone H3K27 methylation, as judged by the intensity of bands of incorporated tritium. The stimulatory effect of full-length huntingtin, compared with reactions without huntingtin or with control peptides, was observed over a range of huntingtin (Supplementary Material, Fig. S4A) and nucleosomal array (Supplementary Material, Fig. S4B) concentrations. Furthermore, consistent with the finding that huntingtin was needed to stimulate tri- but not di-methylation of histone H3K27 in vivo (Fig. 2B, Supplementary Material, Fig. S3B), the results of immunoblot analysis of the in vitro PRC2 reaction products demonstrated that recombinant huntingtin specifically enhanced histone H3K27 tri-methylation but not di-methylation (Supplementary Material, Fig. S4C).

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