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Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes.

Yang C, van der Woerd MJ, Muthurajan UM, Hansen JC, Luger K - Nucleic Acids Res. (2011)

Bottom Line: We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present.MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA.In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Howard Hughes Medical Institute, Colorado State University, Fort Collins, CO 80523-1870, USA.

ABSTRACT
MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

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Analytical ultracentrifugation of MeCP2-nucleosome complexes. (A) Sedimentation velocity analysis of A-Nuc147 and A-Nuc147-MeCP2 complexes in 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT (TCS). Data sets: filled circles A-147Nuc; squares – A-147Nuc-MeCP2 1:1 complex; triangles—A-147Nuc-MeCP2 1:2 complex (B) A-Nuc147-MeCP2 complexes analysed in (A) were run on a 5% native gel after the sedimentation experiment and stained with ethidium bromide. The complexes were still intact after the sedimentation experiments. (C) Sedimentation velocity analysis of W-Nuc165 and W-Nuc165-MeCP2 complexes, under conditions as described in (A). Filled circles: W-Nuc165, squares: W-Nuc165 with an equimolar amount of MeCP2, diamonds: 2-fold molar excess of MeCP2. (D) Ethidium bromide stained 5% native gel of a constant amount of W-Nuc165 titrated with increasing amounts of MeCP2, numbers on the top indicate molar ratios of MeCP2. Asterisks indicate the samples that were analysed by AUC.
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Figure 3: Analytical ultracentrifugation of MeCP2-nucleosome complexes. (A) Sedimentation velocity analysis of A-Nuc147 and A-Nuc147-MeCP2 complexes in 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT (TCS). Data sets: filled circles A-147Nuc; squares – A-147Nuc-MeCP2 1:1 complex; triangles—A-147Nuc-MeCP2 1:2 complex (B) A-Nuc147-MeCP2 complexes analysed in (A) were run on a 5% native gel after the sedimentation experiment and stained with ethidium bromide. The complexes were still intact after the sedimentation experiments. (C) Sedimentation velocity analysis of W-Nuc165 and W-Nuc165-MeCP2 complexes, under conditions as described in (A). Filled circles: W-Nuc165, squares: W-Nuc165 with an equimolar amount of MeCP2, diamonds: 2-fold molar excess of MeCP2. (D) Ethidium bromide stained 5% native gel of a constant amount of W-Nuc165 titrated with increasing amounts of MeCP2, numbers on the top indicate molar ratios of MeCP2. Asterisks indicate the samples that were analysed by AUC.

Mentions: To better understand the structures of the MeCP2-nucleosome complexes identified by EMSA experiments, we employed sedimentation velocity in the analytical ultracentrifuge (SV-AUC). Full length MeCP2 is a monomer in solution with a sedimentation coefficient of 2.3 S and an anomalously high frictional coefficient due to its intrinsically disordered nature (9). The diffusion-corrected sedimentation coefficient distributions of A-Nuc147 alone and in complex with MeCP2 at two different ratios are shown in Figure 3A. Under our experimental conditions, A-Nuc147 sediments as a homogeneous 11 S species, consistent with earlier studies of isolated nucleosome core particles (35). Quite surprisingly, the 1:1.5 and 1:3.0 MeCP2–A-Nuc147 complexes sedimented more slowly than the nucleosome itself (∼10.4 S and ∼10.7 S, respectively). Given that a ratio of 1:1.5 produced a 1:1 MeCP2–A-Nuc147 complex (Supplementary Figure S2; see above) and that the sedimentation coefficient of a di-nucleosome is 15 S (36), the SV-AUC data indicate that the 20% increase in molecular weight due to one MeCP2 binding to a single A-Nuc147 is offset by the increase in frictional coefficient of the resulting MeCP2–A-Nuc147 complex. Importantly, the gel shown in Figure 3B was run after the samples had been subjected to SV-AUC. Thus, we conclude that the increase in electrophoretic shifts obtained upon increasing the MeCP2-nucleosome ratio from 1.5 to 3.0 are due to the interaction of one, then additional MeCP2 molecule(s), with a single A-Nuc147.Figure 3.


Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes.

Yang C, van der Woerd MJ, Muthurajan UM, Hansen JC, Luger K - Nucleic Acids Res. (2011)

Analytical ultracentrifugation of MeCP2-nucleosome complexes. (A) Sedimentation velocity analysis of A-Nuc147 and A-Nuc147-MeCP2 complexes in 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT (TCS). Data sets: filled circles A-147Nuc; squares – A-147Nuc-MeCP2 1:1 complex; triangles—A-147Nuc-MeCP2 1:2 complex (B) A-Nuc147-MeCP2 complexes analysed in (A) were run on a 5% native gel after the sedimentation experiment and stained with ethidium bromide. The complexes were still intact after the sedimentation experiments. (C) Sedimentation velocity analysis of W-Nuc165 and W-Nuc165-MeCP2 complexes, under conditions as described in (A). Filled circles: W-Nuc165, squares: W-Nuc165 with an equimolar amount of MeCP2, diamonds: 2-fold molar excess of MeCP2. (D) Ethidium bromide stained 5% native gel of a constant amount of W-Nuc165 titrated with increasing amounts of MeCP2, numbers on the top indicate molar ratios of MeCP2. Asterisks indicate the samples that were analysed by AUC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105411&req=5

Figure 3: Analytical ultracentrifugation of MeCP2-nucleosome complexes. (A) Sedimentation velocity analysis of A-Nuc147 and A-Nuc147-MeCP2 complexes in 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT (TCS). Data sets: filled circles A-147Nuc; squares – A-147Nuc-MeCP2 1:1 complex; triangles—A-147Nuc-MeCP2 1:2 complex (B) A-Nuc147-MeCP2 complexes analysed in (A) were run on a 5% native gel after the sedimentation experiment and stained with ethidium bromide. The complexes were still intact after the sedimentation experiments. (C) Sedimentation velocity analysis of W-Nuc165 and W-Nuc165-MeCP2 complexes, under conditions as described in (A). Filled circles: W-Nuc165, squares: W-Nuc165 with an equimolar amount of MeCP2, diamonds: 2-fold molar excess of MeCP2. (D) Ethidium bromide stained 5% native gel of a constant amount of W-Nuc165 titrated with increasing amounts of MeCP2, numbers on the top indicate molar ratios of MeCP2. Asterisks indicate the samples that were analysed by AUC.
Mentions: To better understand the structures of the MeCP2-nucleosome complexes identified by EMSA experiments, we employed sedimentation velocity in the analytical ultracentrifuge (SV-AUC). Full length MeCP2 is a monomer in solution with a sedimentation coefficient of 2.3 S and an anomalously high frictional coefficient due to its intrinsically disordered nature (9). The diffusion-corrected sedimentation coefficient distributions of A-Nuc147 alone and in complex with MeCP2 at two different ratios are shown in Figure 3A. Under our experimental conditions, A-Nuc147 sediments as a homogeneous 11 S species, consistent with earlier studies of isolated nucleosome core particles (35). Quite surprisingly, the 1:1.5 and 1:3.0 MeCP2–A-Nuc147 complexes sedimented more slowly than the nucleosome itself (∼10.4 S and ∼10.7 S, respectively). Given that a ratio of 1:1.5 produced a 1:1 MeCP2–A-Nuc147 complex (Supplementary Figure S2; see above) and that the sedimentation coefficient of a di-nucleosome is 15 S (36), the SV-AUC data indicate that the 20% increase in molecular weight due to one MeCP2 binding to a single A-Nuc147 is offset by the increase in frictional coefficient of the resulting MeCP2–A-Nuc147 complex. Importantly, the gel shown in Figure 3B was run after the samples had been subjected to SV-AUC. Thus, we conclude that the increase in electrophoretic shifts obtained upon increasing the MeCP2-nucleosome ratio from 1.5 to 3.0 are due to the interaction of one, then additional MeCP2 molecule(s), with a single A-Nuc147.Figure 3.

Bottom Line: We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present.MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA.In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Howard Hughes Medical Institute, Colorado State University, Fort Collins, CO 80523-1870, USA.

ABSTRACT
MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

Show MeSH
Related in: MedlinePlus