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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 forms defined complexes with nucleosomes in vitro. Gels in (A, B, C and E) were stained in ethidium bromide, the gel in D was stained with Coomassie blue. (A) Increasing amounts of MeCP2 were added to A-Nuc147 resulting in retarded mobility of A-Nuc147. In this preparation, nucleosomes (in absence of MeCP2) migrate as two bands on the gel, representative of two different positions of the histone octamer with respect to the DNA (24). (B) Mobility shifts were also observed with MeCP2 N-terminal fragments (amino acids 1–305 or 78–305), indicating that the N-terminal portion of MeCP2 is sufficient for this interaction. (C and D) Upper and lower bands (C) were excised and eluted from the gel before loading onto an 18% SDS gel (D). (E) Electrophoretic mobility shifts were also observed with W-Nuc165, with both full length and the N-terminal MeCP2 (78–305) fragment.
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Figure 2: MeCP2 forms defined complexes with nucleosomes in vitro. Gels in (A, B, C and E) were stained in ethidium bromide, the gel in D was stained with Coomassie blue. (A) Increasing amounts of MeCP2 were added to A-Nuc147 resulting in retarded mobility of A-Nuc147. In this preparation, nucleosomes (in absence of MeCP2) migrate as two bands on the gel, representative of two different positions of the histone octamer with respect to the DNA (24). (B) Mobility shifts were also observed with MeCP2 N-terminal fragments (amino acids 1–305 or 78–305), indicating that the N-terminal portion of MeCP2 is sufficient for this interaction. (C and D) Upper and lower bands (C) were excised and eluted from the gel before loading onto an 18% SDS gel (D). (E) Electrophoretic mobility shifts were also observed with W-Nuc165, with both full length and the N-terminal MeCP2 (78–305) fragment.

Mentions: Nucleosomes reconstituted with a 147 bp DNA fragment derived from α-sat DNA [A-Nuc-147; (22)] were incubated with increasing amounts of full length human recombinant MeCP2 and the complexes were resolved on a 5% native polyacrylamide gel (Figure 2A). The addition of increasing amounts of MeCP2 resulted in a progressive reduction of free A-Nuc147 and the appearance of discretely shifted and super-shifted bands. Nucleosomes in the off-centered position (Figure 2A, lane 2, upper band) shifted preferentially. The same behavior was observed with RTT MeCP2 fragments 1–305 or 78–305 (which encompasses the MBD, ID and TRD; Figure 2B), as well as with the individual MBD or TRD fragments (data not shown). Two bands containing putative A-Nuc147-MeCP2(1–305) complexes were excised from the native gel (Figure 2C) and loaded onto an 18% SDS gel (Figure 2D). All four histones as well as MeCP2(1–305) were present in both bands. The relative intensities of the four core histones and MeCP2 bands are very similar in the two shifted complexes (Figure 2D, lanes 4 and 5). Because the upper shifted band reverts to the lower shifted band upon addition of more nucleosomes (data not shown), we interpret the upper super-shifted complex as having multiple MeCP2 molecules bound to one nucleosome. Similar MeCP2-nucleosome complexes were obtained after incubation in the presence of 150 and 300 mM NaCl (data not shown). Nucleosomes reconstituted with a 165 bp DNA fragment centered on a strong nucleosome positioning sequence [‘601’; (23); W-Nuc165] exhibited the same behaviour in EMSAs as the linker-less A-Nuc147 nucleosomes in the presence of full length and truncated MeCP2 (Figure 2E).Figure 2.


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 forms defined complexes with nucleosomes in vitro. Gels in (A, B, C and E) were stained in ethidium bromide, the gel in D was stained with Coomassie blue. (A) Increasing amounts of MeCP2 were added to A-Nuc147 resulting in retarded mobility of A-Nuc147. In this preparation, nucleosomes (in absence of MeCP2) migrate as two bands on the gel, representative of two different positions of the histone octamer with respect to the DNA (24). (B) Mobility shifts were also observed with MeCP2 N-terminal fragments (amino acids 1–305 or 78–305), indicating that the N-terminal portion of MeCP2 is sufficient for this interaction. (C and D) Upper and lower bands (C) were excised and eluted from the gel before loading onto an 18% SDS gel (D). (E) Electrophoretic mobility shifts were also observed with W-Nuc165, with both full length and the N-terminal MeCP2 (78–305) fragment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: MeCP2 forms defined complexes with nucleosomes in vitro. Gels in (A, B, C and E) were stained in ethidium bromide, the gel in D was stained with Coomassie blue. (A) Increasing amounts of MeCP2 were added to A-Nuc147 resulting in retarded mobility of A-Nuc147. In this preparation, nucleosomes (in absence of MeCP2) migrate as two bands on the gel, representative of two different positions of the histone octamer with respect to the DNA (24). (B) Mobility shifts were also observed with MeCP2 N-terminal fragments (amino acids 1–305 or 78–305), indicating that the N-terminal portion of MeCP2 is sufficient for this interaction. (C and D) Upper and lower bands (C) were excised and eluted from the gel before loading onto an 18% SDS gel (D). (E) Electrophoretic mobility shifts were also observed with W-Nuc165, with both full length and the N-terminal MeCP2 (78–305) fragment.
Mentions: Nucleosomes reconstituted with a 147 bp DNA fragment derived from α-sat DNA [A-Nuc-147; (22)] were incubated with increasing amounts of full length human recombinant MeCP2 and the complexes were resolved on a 5% native polyacrylamide gel (Figure 2A). The addition of increasing amounts of MeCP2 resulted in a progressive reduction of free A-Nuc147 and the appearance of discretely shifted and super-shifted bands. Nucleosomes in the off-centered position (Figure 2A, lane 2, upper band) shifted preferentially. The same behavior was observed with RTT MeCP2 fragments 1–305 or 78–305 (which encompasses the MBD, ID and TRD; Figure 2B), as well as with the individual MBD or TRD fragments (data not shown). Two bands containing putative A-Nuc147-MeCP2(1–305) complexes were excised from the native gel (Figure 2C) and loaded onto an 18% SDS gel (Figure 2D). All four histones as well as MeCP2(1–305) were present in both bands. The relative intensities of the four core histones and MeCP2 bands are very similar in the two shifted complexes (Figure 2D, lanes 4 and 5). Because the upper shifted band reverts to the lower shifted band upon addition of more nucleosomes (data not shown), we interpret the upper super-shifted complex as having multiple MeCP2 molecules bound to one nucleosome. Similar MeCP2-nucleosome complexes were obtained after incubation in the presence of 150 and 300 mM NaCl (data not shown). Nucleosomes reconstituted with a 165 bp DNA fragment centered on a strong nucleosome positioning sequence [‘601’; (23); W-Nuc165] exhibited the same behaviour in EMSAs as the linker-less A-Nuc147 nucleosomes in the presence of full length and truncated MeCP2 (Figure 2E).Figure 2.

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