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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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Models for MeCP2–nucleosome interactions: nucleosomes and MeCP2 are shown to scale, according to the dmax values determined by SAXS (Table 2). Note that dmax for highly extended proteins such as MeCP2 is notoriously difficult to determine. The model takes into account that MeCP2 binds near the nucleosomal dyad. (A) W-Nuc146 interacts with multiple copies of full length MeCP2 (as judged from Porod volumes; Table 2) to form extended complexes. (B) A-Nuc147 interacts with a single copy of MeCP2 or MeCP278–305 (as shown by SEC-MALS, Supplementary Figure S2), (C) W-Nuc165 also forms defined 1:1 complexes with both versions of MeCP2. The relatively minor changes in dmax between unbound nucleosomes and those in complex with full length MeCP2 or MeCP278–305 suggests that DNA binding domains in the C-terminal region of MeCP2 are also engaged in interactions with this nucleosome.
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Figure 8: Models for MeCP2–nucleosome interactions: nucleosomes and MeCP2 are shown to scale, according to the dmax values determined by SAXS (Table 2). Note that dmax for highly extended proteins such as MeCP2 is notoriously difficult to determine. The model takes into account that MeCP2 binds near the nucleosomal dyad. (A) W-Nuc146 interacts with multiple copies of full length MeCP2 (as judged from Porod volumes; Table 2) to form extended complexes. (B) A-Nuc147 interacts with a single copy of MeCP2 or MeCP278–305 (as shown by SEC-MALS, Supplementary Figure S2), (C) W-Nuc165 also forms defined 1:1 complexes with both versions of MeCP2. The relatively minor changes in dmax between unbound nucleosomes and those in complex with full length MeCP2 or MeCP278–305 suggests that DNA binding domains in the C-terminal region of MeCP2 are also engaged in interactions with this nucleosome.

Mentions: MeCP2 is an intrinsically disordered protein for which only limited structural information exists. Here, we present the biophysical and structural characterization of several MeCP2-nucleosome complexes. We demonstrate that one molecule of MeCP2 forms a well-defined complex with nucleosomes with and without extra-nucleosomal linker DNA, without displacing any of the core histones. The molecular dimensions obtained from SAXS experiments demonstrate that MeCP2, which is extended and disordered in its free state, assumes a less extended conformation upon interacting with nucleosomes containing linker DNA, but remains extended when interacting with nucleosomes without DNA linkers. This suggests that additional DNA binding domains in MeCP2 become engaged upon the interaction with linker DNA and nucleosomes. Together, our data allow us to build a working model for MeCP2-nucleosome interactions (Figure 8). Finally, SAXS experiments reveal unexpected differences in DNA end conformations between nucleosomes reconstituted with two different DNA sequences.Figure 8.


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)

Models for MeCP2–nucleosome interactions: nucleosomes and MeCP2 are shown to scale, according to the dmax values determined by SAXS (Table 2). Note that dmax for highly extended proteins such as MeCP2 is notoriously difficult to determine. The model takes into account that MeCP2 binds near the nucleosomal dyad. (A) W-Nuc146 interacts with multiple copies of full length MeCP2 (as judged from Porod volumes; Table 2) to form extended complexes. (B) A-Nuc147 interacts with a single copy of MeCP2 or MeCP278–305 (as shown by SEC-MALS, Supplementary Figure S2), (C) W-Nuc165 also forms defined 1:1 complexes with both versions of MeCP2. The relatively minor changes in dmax between unbound nucleosomes and those in complex with full length MeCP2 or MeCP278–305 suggests that DNA binding domains in the C-terminal region of MeCP2 are also engaged in interactions with this nucleosome.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Models for MeCP2–nucleosome interactions: nucleosomes and MeCP2 are shown to scale, according to the dmax values determined by SAXS (Table 2). Note that dmax for highly extended proteins such as MeCP2 is notoriously difficult to determine. The model takes into account that MeCP2 binds near the nucleosomal dyad. (A) W-Nuc146 interacts with multiple copies of full length MeCP2 (as judged from Porod volumes; Table 2) to form extended complexes. (B) A-Nuc147 interacts with a single copy of MeCP2 or MeCP278–305 (as shown by SEC-MALS, Supplementary Figure S2), (C) W-Nuc165 also forms defined 1:1 complexes with both versions of MeCP2. The relatively minor changes in dmax between unbound nucleosomes and those in complex with full length MeCP2 or MeCP278–305 suggests that DNA binding domains in the C-terminal region of MeCP2 are also engaged in interactions with this nucleosome.
Mentions: MeCP2 is an intrinsically disordered protein for which only limited structural information exists. Here, we present the biophysical and structural characterization of several MeCP2-nucleosome complexes. We demonstrate that one molecule of MeCP2 forms a well-defined complex with nucleosomes with and without extra-nucleosomal linker DNA, without displacing any of the core histones. The molecular dimensions obtained from SAXS experiments demonstrate that MeCP2, which is extended and disordered in its free state, assumes a less extended conformation upon interacting with nucleosomes containing linker DNA, but remains extended when interacting with nucleosomes without DNA linkers. This suggests that additional DNA binding domains in MeCP2 become engaged upon the interaction with linker DNA and nucleosomes. Together, our data allow us to build a working model for MeCP2-nucleosome interactions (Figure 8). Finally, SAXS experiments reveal unexpected differences in DNA end conformations between nucleosomes reconstituted with two different DNA sequences.Figure 8.

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