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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 analysis of W-Nuc146 as a proof of concept. (A) The crystal structure of the nucleosome core particle [PDB entry 1AOI, (22)], which consists of two copies each of H2A (yellow), H2B (red), H3 (blue), H4 (green) and 147 bp of DNA (gray), with the histone tails removed, serves as a reference. The particle measures approximately 100 × 100 × 60 Å in size. To allow comparison of particle sizes, these size indicators are consistently used in envelope reconstructions in this paper. (B) Molecular envelope derived from SAXS data for W-Nuc146. This envelope is an average of ten reconstructions, acquired with the program DAMMIN (29) in slow mode. Movies of these envelopes are available in Supplementary Data, giving a better appreciation of three-dimensional views of these envelopes. (C) (Left panel) Experimental scattering data (red) superimposed with simulated data from the crystal structure (black), generated with the program CRYSOL (41) with PDB entry 1AOI as input (histone tails removed from the model). The CRYSOL χ value for the match of the crystal structure with the experimental data is shown in the figure (value in parentheses). (Right panel) The same data after inversion to P(r) functions by the program AUTOGNOM (28). Both graphs show that the crystal structure is an excellent model to explain the experimental scattering data.
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Figure 5: SAXS analysis of W-Nuc146 as a proof of concept. (A) The crystal structure of the nucleosome core particle [PDB entry 1AOI, (22)], which consists of two copies each of H2A (yellow), H2B (red), H3 (blue), H4 (green) and 147 bp of DNA (gray), with the histone tails removed, serves as a reference. The particle measures approximately 100 × 100 × 60 Å in size. To allow comparison of particle sizes, these size indicators are consistently used in envelope reconstructions in this paper. (B) Molecular envelope derived from SAXS data for W-Nuc146. This envelope is an average of ten reconstructions, acquired with the program DAMMIN (29) in slow mode. Movies of these envelopes are available in Supplementary Data, giving a better appreciation of three-dimensional views of these envelopes. (C) (Left panel) Experimental scattering data (red) superimposed with simulated data from the crystal structure (black), generated with the program CRYSOL (41) with PDB entry 1AOI as input (histone tails removed from the model). The CRYSOL χ value for the match of the crystal structure with the experimental data is shown in the figure (value in parentheses). (Right panel) The same data after inversion to P(r) functions by the program AUTOGNOM (28). Both graphs show that the crystal structure is an excellent model to explain the experimental scattering data.

Mentions: SAXS provides information on the dimensions and shape of macromolecules in solution. Additionally, structural information can be calculated from SAXS data at 10–50 Å resolution (37,38). We used SAXS to obtain complementary information on nucleosome-MeCP2 complexes. As an important control, we first obtained scattering data from nucleosomes in the absence of MeCP2. While the crystal structure of A-Nuc147 is known to a very high resolution (39) and the structures of W-Nuc146 to medium resolution (40), the structure for W-Nuc165 is not known. The published structures of A-Nuc147 and W-Nuc146 are very similar: both structures show approximately 1.75 turns of double stranded DNA wound around the histone octamer. Figure 5A (based on the A-Nuc147 structure) is representative for both crystal structures. To allow for a direct comparison between nucleosomes with and without linker DNA and to test whether DNA sequence had an effect on overall nucleosome structure in solution, we performed SAXS on all three nucleosomes. The same samples that were used for SAXS were shown to be highly homogeneous using EMSA and by SEC-MALS (Supplementary Figure S2).Figure 5.


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 analysis of W-Nuc146 as a proof of concept. (A) The crystal structure of the nucleosome core particle [PDB entry 1AOI, (22)], which consists of two copies each of H2A (yellow), H2B (red), H3 (blue), H4 (green) and 147 bp of DNA (gray), with the histone tails removed, serves as a reference. The particle measures approximately 100 × 100 × 60 Å in size. To allow comparison of particle sizes, these size indicators are consistently used in envelope reconstructions in this paper. (B) Molecular envelope derived from SAXS data for W-Nuc146. This envelope is an average of ten reconstructions, acquired with the program DAMMIN (29) in slow mode. Movies of these envelopes are available in Supplementary Data, giving a better appreciation of three-dimensional views of these envelopes. (C) (Left panel) Experimental scattering data (red) superimposed with simulated data from the crystal structure (black), generated with the program CRYSOL (41) with PDB entry 1AOI as input (histone tails removed from the model). The CRYSOL χ value for the match of the crystal structure with the experimental data is shown in the figure (value in parentheses). (Right panel) The same data after inversion to P(r) functions by the program AUTOGNOM (28). Both graphs show that the crystal structure is an excellent model to explain the experimental scattering data.
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Related In: Results  -  Collection

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Figure 5: SAXS analysis of W-Nuc146 as a proof of concept. (A) The crystal structure of the nucleosome core particle [PDB entry 1AOI, (22)], which consists of two copies each of H2A (yellow), H2B (red), H3 (blue), H4 (green) and 147 bp of DNA (gray), with the histone tails removed, serves as a reference. The particle measures approximately 100 × 100 × 60 Å in size. To allow comparison of particle sizes, these size indicators are consistently used in envelope reconstructions in this paper. (B) Molecular envelope derived from SAXS data for W-Nuc146. This envelope is an average of ten reconstructions, acquired with the program DAMMIN (29) in slow mode. Movies of these envelopes are available in Supplementary Data, giving a better appreciation of three-dimensional views of these envelopes. (C) (Left panel) Experimental scattering data (red) superimposed with simulated data from the crystal structure (black), generated with the program CRYSOL (41) with PDB entry 1AOI as input (histone tails removed from the model). The CRYSOL χ value for the match of the crystal structure with the experimental data is shown in the figure (value in parentheses). (Right panel) The same data after inversion to P(r) functions by the program AUTOGNOM (28). Both graphs show that the crystal structure is an excellent model to explain the experimental scattering data.
Mentions: SAXS provides information on the dimensions and shape of macromolecules in solution. Additionally, structural information can be calculated from SAXS data at 10–50 Å resolution (37,38). We used SAXS to obtain complementary information on nucleosome-MeCP2 complexes. As an important control, we first obtained scattering data from nucleosomes in the absence of MeCP2. While the crystal structure of A-Nuc147 is known to a very high resolution (39) and the structures of W-Nuc146 to medium resolution (40), the structure for W-Nuc165 is not known. The published structures of A-Nuc147 and W-Nuc146 are very similar: both structures show approximately 1.75 turns of double stranded DNA wound around the histone octamer. Figure 5A (based on the A-Nuc147 structure) is representative for both crystal structures. To allow for a direct comparison between nucleosomes with and without linker DNA and to test whether DNA sequence had an effect on overall nucleosome structure in solution, we performed SAXS on all three nucleosomes. The same samples that were used for SAXS were shown to be highly homogeneous using EMSA and by SEC-MALS (Supplementary Figure S2).Figure 5.

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