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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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MeCP2 preferentially interacts with nucleosomes with extra-nucleosomal linker DNA. (A) MeCP2 was pre-incubated with W-Nuc146, and an increasing amount of fluorescently labeled W-Nuc165 (W-Nuc165*) was added as a competitor (lanes 4–6). In lanes 8–10, W-Nuc146 was added as competitor to pre-incubated W-Nuc165*-MeCP2 complex. (B) Fluorescence view of the gel shown in (A), demonstrating the increase in fluorescently labeled 165NCP-MeCP2 complex when W-Nuc165* was added as a competitor (lanes 4–6), but no significant change was observed in lanes 9, 10 when W-146Nuc was added as a competitor. (C) Competition assay between 53 bp DNA and W-Nuc165. 53mer DNA was added to pre-incubated W-Nuc165*-MeCP2 complex, resulting in the formation of MeCP2-DNA complexes and free nucleosome (lanes 8–10).
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Figure 4: MeCP2 preferentially interacts with nucleosomes with extra-nucleosomal linker DNA. (A) MeCP2 was pre-incubated with W-Nuc146, and an increasing amount of fluorescently labeled W-Nuc165 (W-Nuc165*) was added as a competitor (lanes 4–6). In lanes 8–10, W-Nuc146 was added as competitor to pre-incubated W-Nuc165*-MeCP2 complex. (B) Fluorescence view of the gel shown in (A), demonstrating the increase in fluorescently labeled 165NCP-MeCP2 complex when W-Nuc165* was added as a competitor (lanes 4–6), but no significant change was observed in lanes 9, 10 when W-146Nuc was added as a competitor. (C) Competition assay between 53 bp DNA and W-Nuc165. 53mer DNA was added to pre-incubated W-Nuc165*-MeCP2 complex, resulting in the formation of MeCP2-DNA complexes and free nucleosome (lanes 8–10).

Mentions: Previous quantitative studies have observed robust interaction with nucleosomes assembled on longer DNA fragments containing linker DNA (11,20). To differentiate the relative binding affinities of MeCP2 for nucleosomes with and without linker DNA, we used a competition assay. To exclude possible sequence-dependent differences between the two nucleosomes and to directly investigate the effect of linker DNA on binding affinity, we prepared nucleosomes with the central 146 bp of the 601 sequence used for W-Nuc165 (W-Nuc146; Supplementary Figure S1). W-Nuc146 was pre-incubated with full length MeCP2 at a molar ratio ranging from 1 to 1.5. Increasing amounts of fluorescently labeled W-Nuc165 was added and incubated at room temperature for another 30 min before resolving the complexes by 5% native PAGE. Fluorescently labeled W-Nuc165 successfully competes with W-Nuc146 for MeCP2, as observed in an increase in the amount of W-Nuc165–MeCP2 complex, accompanied by an increase of free W-Nuc146 (Figure 4A, lanes 3–6). This is particularly apparent when only fluorescently labeled W-Nuc165 is visualized (Figure 4B, lanes 4–6). At lower concentrations, all of the W-Nuc165 is found in complex with MeCP2. Conversely, when increasing amounts of W-Nuc146 were added as a competitor to the pre-formed W-Nuc165-MeCP2 complex, the amount of 165NCP-MeCP2 complexes remains nearly unchanged (Figure 4A and B, lanes 8–10).Figure 4.


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)

MeCP2 preferentially interacts with nucleosomes with extra-nucleosomal linker DNA. (A) MeCP2 was pre-incubated with W-Nuc146, and an increasing amount of fluorescently labeled W-Nuc165 (W-Nuc165*) was added as a competitor (lanes 4–6). In lanes 8–10, W-Nuc146 was added as competitor to pre-incubated W-Nuc165*-MeCP2 complex. (B) Fluorescence view of the gel shown in (A), demonstrating the increase in fluorescently labeled 165NCP-MeCP2 complex when W-Nuc165* was added as a competitor (lanes 4–6), but no significant change was observed in lanes 9, 10 when W-146Nuc was added as a competitor. (C) Competition assay between 53 bp DNA and W-Nuc165. 53mer DNA was added to pre-incubated W-Nuc165*-MeCP2 complex, resulting in the formation of MeCP2-DNA complexes and free nucleosome (lanes 8–10).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105411&req=5

Figure 4: MeCP2 preferentially interacts with nucleosomes with extra-nucleosomal linker DNA. (A) MeCP2 was pre-incubated with W-Nuc146, and an increasing amount of fluorescently labeled W-Nuc165 (W-Nuc165*) was added as a competitor (lanes 4–6). In lanes 8–10, W-Nuc146 was added as competitor to pre-incubated W-Nuc165*-MeCP2 complex. (B) Fluorescence view of the gel shown in (A), demonstrating the increase in fluorescently labeled 165NCP-MeCP2 complex when W-Nuc165* was added as a competitor (lanes 4–6), but no significant change was observed in lanes 9, 10 when W-146Nuc was added as a competitor. (C) Competition assay between 53 bp DNA and W-Nuc165. 53mer DNA was added to pre-incubated W-Nuc165*-MeCP2 complex, resulting in the formation of MeCP2-DNA complexes and free nucleosome (lanes 8–10).
Mentions: Previous quantitative studies have observed robust interaction with nucleosomes assembled on longer DNA fragments containing linker DNA (11,20). To differentiate the relative binding affinities of MeCP2 for nucleosomes with and without linker DNA, we used a competition assay. To exclude possible sequence-dependent differences between the two nucleosomes and to directly investigate the effect of linker DNA on binding affinity, we prepared nucleosomes with the central 146 bp of the 601 sequence used for W-Nuc165 (W-Nuc146; Supplementary Figure S1). W-Nuc146 was pre-incubated with full length MeCP2 at a molar ratio ranging from 1 to 1.5. Increasing amounts of fluorescently labeled W-Nuc165 was added and incubated at room temperature for another 30 min before resolving the complexes by 5% native PAGE. Fluorescently labeled W-Nuc165 successfully competes with W-Nuc146 for MeCP2, as observed in an increase in the amount of W-Nuc165–MeCP2 complex, accompanied by an increase of free W-Nuc146 (Figure 4A, lanes 3–6). This is particularly apparent when only fluorescently labeled W-Nuc165 is visualized (Figure 4B, lanes 4–6). At lower concentrations, all of the W-Nuc165 is found in complex with MeCP2. Conversely, when increasing amounts of W-Nuc146 were added as a competitor to the pre-formed W-Nuc165-MeCP2 complex, the amount of 165NCP-MeCP2 complexes remains nearly unchanged (Figure 4A and B, lanes 8–10).Figure 4.

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