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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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SAXS reveals sequence-dependent conformational heterogeneity in nucleosomes. (A) The average molecular envelope reconstructed from SAXS data for A-Nuc147. It is distinctly different from the envelope shown in Figure 5B and has a less round character. (B.1) The experimental scattering data for A-Nuc147 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for A-Nuc147 (blue) are superimposed. The CRYSOL χ values for the match of the respective models with the experimental data are shown in parentheses in the figure. A cartoon of a new model for A-Nuc147 is shown in the inset. The model was made by rearranging the terminal six DNA base pairs on each end. (B.2) Experimental scattering data for W-Nuc165 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for W-Nuc165 (blue) are superimposed. CRYSOL χ values are shown for each model. A cartoon of the model is shown in the inset. The model was made by extending the DNA by 7 and 11 bp, respectively. (C) Average molecular envelope reconstructed for W-165Nuc.
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Figure 6: SAXS reveals sequence-dependent conformational heterogeneity in nucleosomes. (A) The average molecular envelope reconstructed from SAXS data for A-Nuc147. It is distinctly different from the envelope shown in Figure 5B and has a less round character. (B.1) The experimental scattering data for A-Nuc147 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for A-Nuc147 (blue) are superimposed. The CRYSOL χ values for the match of the respective models with the experimental data are shown in parentheses in the figure. A cartoon of a new model for A-Nuc147 is shown in the inset. The model was made by rearranging the terminal six DNA base pairs on each end. (B.2) Experimental scattering data for W-Nuc165 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for W-Nuc165 (blue) are superimposed. CRYSOL χ values are shown for each model. A cartoon of the model is shown in the inset. The model was made by extending the DNA by 7 and 11 bp, respectively. (C) Average molecular envelope reconstructed for W-165Nuc.

Mentions: A-Nuc147 (with α-sat DNA) is slightly larger (dmax of 145 Å; Table 2), indicating differences compared with the corresponding crystal structure. This is also obvious from the comparison of the experimental scattering curve with a scattering curve calculated from the crystal structure (Figure 6B.1). Envelopes calculated from the experimental data show a shape that does not correspond to the crystal structure (compare Figure 6A with Figure 5A). Preparations were carefully checked with native PAGE and SEC-MALS to exclude the possibility of contaminants and inhomogeneity, respectively, and similar results have been obtained from three independent nucleosome preparations. We interpret this changed shape by partial unpeeling of nucleosomal DNA from the histone octamer in at least a significant fraction of the nucleosomes in solution. In forward calculations, a nucleosome with 6–10 bp of DNA unpeeled from the body of the histone octamer gave a much better match to the experimental intensity curve (Figure 6B.1). Furthermore, this interpretation is supported by increased sensitivity of the DNA ends towards micrococcal nuclease digestion compared with W-Nuc146 (Supplementary Figure S4).Figure 6.


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)

SAXS reveals sequence-dependent conformational heterogeneity in nucleosomes. (A) The average molecular envelope reconstructed from SAXS data for A-Nuc147. It is distinctly different from the envelope shown in Figure 5B and has a less round character. (B.1) The experimental scattering data for A-Nuc147 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for A-Nuc147 (blue) are superimposed. The CRYSOL χ values for the match of the respective models with the experimental data are shown in parentheses in the figure. A cartoon of a new model for A-Nuc147 is shown in the inset. The model was made by rearranging the terminal six DNA base pairs on each end. (B.2) Experimental scattering data for W-Nuc165 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for W-Nuc165 (blue) are superimposed. CRYSOL χ values are shown for each model. A cartoon of the model is shown in the inset. The model was made by extending the DNA by 7 and 11 bp, respectively. (C) Average molecular envelope reconstructed for W-165Nuc.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: SAXS reveals sequence-dependent conformational heterogeneity in nucleosomes. (A) The average molecular envelope reconstructed from SAXS data for A-Nuc147. It is distinctly different from the envelope shown in Figure 5B and has a less round character. (B.1) The experimental scattering data for A-Nuc147 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for A-Nuc147 (blue) are superimposed. The CRYSOL χ values for the match of the respective models with the experimental data are shown in parentheses in the figure. A cartoon of a new model for A-Nuc147 is shown in the inset. The model was made by rearranging the terminal six DNA base pairs on each end. (B.2) Experimental scattering data for W-Nuc165 (red), simulated data from the crystal structure 1AOI (histone tails removed, black) and simulated data from a model for W-Nuc165 (blue) are superimposed. CRYSOL χ values are shown for each model. A cartoon of the model is shown in the inset. The model was made by extending the DNA by 7 and 11 bp, respectively. (C) Average molecular envelope reconstructed for W-165Nuc.
Mentions: A-Nuc147 (with α-sat DNA) is slightly larger (dmax of 145 Å; Table 2), indicating differences compared with the corresponding crystal structure. This is also obvious from the comparison of the experimental scattering curve with a scattering curve calculated from the crystal structure (Figure 6B.1). Envelopes calculated from the experimental data show a shape that does not correspond to the crystal structure (compare Figure 6A with Figure 5A). Preparations were carefully checked with native PAGE and SEC-MALS to exclude the possibility of contaminants and inhomogeneity, respectively, and similar results have been obtained from three independent nucleosome preparations. We interpret this changed shape by partial unpeeling of nucleosomal DNA from the histone octamer in at least a significant fraction of the nucleosomes in solution. In forward calculations, a nucleosome with 6–10 bp of DNA unpeeled from the body of the histone octamer gave a much better match to the experimental intensity curve (Figure 6B.1). Furthermore, this interpretation is supported by increased sensitivity of the DNA ends towards micrococcal nuclease digestion compared with W-Nuc146 (Supplementary Figure S4).Figure 6.

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