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Cited by 305Genomics and Chromatin DynamicsEpigenetics and DNA MethylationGenetics and Neurodevelopmental Disorders

The DOI matches its Crossref record, with a small difference in title or year; 39 of the 39 references it cites were found in a registry or an index.

Chromatin Compaction by Human MeCP2

Open access
Georgel, Philippe T., Horowitz-Scherer, Rachel A., Adkins, Nick, Woodcock, Christopher L., Wade, Paul A., Hansen, Jeffrey C.·2003·Journal of Biological Chemistry
DOI 10.1074/jbc.m305308200
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Abstract

MeCP2 is a transcriptional repressor that contains an N-terminal methylated DNA-binding domain, a central transcription regulation domain, and a C-terminal domain of unknown function. Whereas current models of MeCP2 function evoke localized recruitment of histone deacetylases to specific methylated regions of the genome, it is unclear whether MeCP2 requires DNA methylation to bind to chromatin or whether MeCP2 binding influences chromatin structure in the absence of other proteins. To address these issues, we have characterized the complexes formed between MeCP2 and biochemically defined nucleosomal arrays. At molar ratios near 1 MeCP2/nucleosome, unmethylated nucleosomal arrays formed both extensively condensed ellipsoidal particles and oligomeric suprastructures. Furthermore, MeCP2-mediated chromatin compaction occurred in the absence of monovalent or divalent cations, in distinct contrast to all other known chromatin-condensing proteins. Analysis of specific missense and nonsense MeCP2 mutants indicated that the ability to condense chromatin resides in region(s) of the protein other than the methylated DNA-binding domain. These data demonstrate that MeCP2 assembles novel secondary chromatin structures independent of DNA modification and suggest that the ability of MeCP2 to silence chromatin may be related in part to its effects on large-scale chromatin organization.

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Crossref · 10.1074/jbc.m305308200

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Figures

FIG. 1 MeCP2 cross-links naked DNA into large nucleoprotein particles. A , SDS-PAGE of purified human recombinant MeCP2 ( rMeCP2 ). B , native agarose gel electrophoresis. Unmethylated 208-12 DNA ( second through sixth lanes ) was incubated with MeCP2 at the indicated r MeCP2 , and samples were electrophoresed on a 1% agarose gel (1 × Tris acetate/EDTA) as described under ' Experimental Procedures. ' The first lane contains 1-kb + DNA size markers (Invitrogen). The seventh lane contains mock-incubated (no MeCP2) 208-12 DNA. C , EM. MeCP2 and 208-12 DNA were incubated at r MeCP2 = 1.0 and processed for dark-field EM as described under ' Experimental Procedures. ' Panel i , a DNA strand with no apparent bound MeCP2; panels ii -iv , examples of two strands brought into close apposition by bound MeCP2, with places where the two strands diverge indicated by arrows ; panel v , a more complex structure containing several DNA molecules, with two apposed regions indicated by arrows .
FIG. 1 MeCP2 cross-links naked DNA into large nucleoprotein particles. A , SDS-PAGE of purified human recombinant MeCP2 ( rMeCP2 ). B , native agarose gel electrophoresis. Unmethylated 208-12 DNA ( second through sixth lanes ) was incubated with MeCP2 at the indicated r MeCP2 , and samples were electrophoresed on a 1% agarose gel (1 × Tris acetate/EDTA) as described under ' Experimental Procedures. ' The first lane contains 1-kb + DNA size markers (Invitrogen). The seventh lane contains mock-incubated (no MeCP2) 208-12 DNA. C , EM. MeCP2 and 208-12 DNA were incubated at r MeCP2 = 1.0 and processed for dark-field EM as described under ' Experimental Procedures. ' Panel i , a DNA strand with no apparent bound MeCP2; panels ii -iv , examples of two strands brought into close apposition by bound MeCP2, with places where the two strands diverge indicated by arrows ; panel v , a more complex structure containing several DNA molecules, with two apposed regions indicated by arrows .CC-BY · From the PDF
FIG. 2 MeCP2 mediates extensive compaction of nucleosomal arrays. A , native agarose gel electrophoresis. 208-12 nucleosomal arrays ( second through sixth lanes ) were incubated with MeCP2 at the indicated r MeCP2 , and samples were electrophoresed on a 1% agarose gel (1 × Tris acetate/EDTA) as described under ' Experimental Procedures. ' The first lane contains 1-kb + DNA size markers (Invitrogen).The seventh lane contains mock-incubated (no MeCP2) 208-12 nucleosomal arrays. B , shadowed EM. Panel a , an extended 208-12 nucleosomal array loaded with 10 nucleosomes in low salt HEGN buffer. Panels b -f , 208-12 nucleosomal arrays incubated with MeCP2 in HEGN buffer at r MeCP2 = 0.5. An oligomer present under these conditions is shown in panel f . Panels g -i , 208-12 nucleosomal arrays incubated with MeCP2 in HEGN buffer at r MeCP2 = 1.0. Oligomers are again present ( arrows in panel i ). The length of the shadows ( arrowheads in panels g and h ) demonstrates that drying from glycerol in high vacuum retains the three-dimensional structure and avoids flattening.
FIG. 2 MeCP2 mediates extensive compaction of nucleosomal arrays. A , native agarose gel electrophoresis. 208-12 nucleosomal arrays ( second through sixth lanes ) were incubated with MeCP2 at the indicated r MeCP2 , and samples were electrophoresed on a 1% agarose gel (1 × Tris acetate/EDTA) as described under ' Experimental Procedures. ' The first lane contains 1-kb + DNA size markers (Invitrogen).The seventh lane contains mock-incubated (no MeCP2) 208-12 nucleosomal arrays. B , shadowed EM. Panel a , an extended 208-12 nucleosomal array loaded with 10 nucleosomes in low salt HEGN buffer. Panels b -f , 208-12 nucleosomal arrays incubated with MeCP2 in HEGN buffer at r MeCP2 = 0.5. An oligomer present under these conditions is shown in panel f . Panels g -i , 208-12 nucleosomal arrays incubated with MeCP2 in HEGN buffer at r MeCP2 = 1.0. Oligomers are again present ( arrows in panel i ). The length of the shadows ( arrowheads in panels g and h ) demonstrates that drying from glycerol in high vacuum retains the three-dimensional structure and avoids flattening.CC-BY · From the PDF
Image 3
CC-BY · From the PDF
FIG. 4 Three-dimensional reconstructions of MeCP2-bound nucleosomal arrays. 208-12 nucleosomal arrays were incubated with MeCP2 at r MeCP2 = 1.0 and visualized by EM in negative stain. Panel a shows single compacted arrays ( black arrow ) and oligomers ( white arrow ) ( bar = 50 nm). Central slices from tomographic reconstructions of individual compacted arrays are shown in panels b and e . The individual arrays in panels b and e were components of the oligomer shown in the inset of Fig. 6. Nucleosome positions and orientations are shown as 5-nm × 10-nm disks. Panels d and g are views of panels c and f , respectively, that have been rotated 90 ° about the vertical axis of the model. Panel h shows ECM images of frozen hydrated samples recorded as stereo pairs ( bar = 50 nm). Single arrays ( black arrows ) and oligomers ( white arrow ) were observed. Three-dimensional locations of nucleosomes in the single arrays ( black arrows in panel h ) were determined without orientation information and are modeled as 10-nm diameter spheres ( panels i -l ). Panels j and l are views of panels i and k , respectively, that have been rotated by 90 o on the vertical axis of the model. The bar in the lower right-hand corner equals 10 nm and applies to the models shown in panels i -l .
FIG. 4 Three-dimensional reconstructions of MeCP2-bound nucleosomal arrays. 208-12 nucleosomal arrays were incubated with MeCP2 at r MeCP2 = 1.0 and visualized by EM in negative stain. Panel a shows single compacted arrays ( black arrow ) and oligomers ( white arrow ) ( bar = 50 nm). Central slices from tomographic reconstructions of individual compacted arrays are shown in panels b and e . The individual arrays in panels b and e were components of the oligomer shown in the inset of Fig. 6. Nucleosome positions and orientations are shown as 5-nm × 10-nm disks. Panels d and g are views of panels c and f , respectively, that have been rotated 90 ° about the vertical axis of the model. Panel h shows ECM images of frozen hydrated samples recorded as stereo pairs ( bar = 50 nm). Single arrays ( black arrows ) and oligomers ( white arrow ) were observed. Three-dimensional locations of nucleosomes in the single arrays ( black arrows in panel h ) were determined without orientation information and are modeled as 10-nm diameter spheres ( panels i -l ). Panels j and l are views of panels i and k , respectively, that have been rotated by 90 o on the vertical axis of the model. The bar in the lower right-hand corner equals 10 nm and applies to the models shown in panels i -l .CC-BY · From the PDF
FIG. 5 Restriction digestion of MeCP2-bound nucleosomal arrays. A , accessibility of linker DNA. Aliquots of either naked 208-12 DNA ( lanes 3 -6 ) or 208-12 nucleosomal arrays incubated with MeCP2 at the indicated r MeCP2 ( lanes 7 -10 ) were digested with Eco RI at 37 ° C for 90 min, phenol/chloroform-extracted to remove bound proteins, and ethanol-precipitated. Resuspended DNA was subsequently electrophoresed on a 1% agarose gel (1 × Tris acetate/EDTA) as described under ' Experimental Procedures. ' Lanes 1 in A and B contain 1-kb + DNA size markers (Invitrogen). B , stability of native complexes after digestion. Aliquots of 208-12 nucleosomal arrays were incubated with MeCP2 at the indicated r MeCP2 , digested with Eco RI at 37 ° Cfor 90 min, and electrophoresed as described for A . Arrowheads indicate the sizes of selected markers in base pairs.
FIG. 5 Restriction digestion of MeCP2-bound nucleosomal arrays. A , accessibility of linker DNA. Aliquots of either naked 208-12 DNA ( lanes 3 -6 ) or 208-12 nucleosomal arrays incubated with MeCP2 at the indicated r MeCP2 ( lanes 7 -10 ) were digested with Eco RI at 37 ° C for 90 min, phenol/chloroform-extracted to remove bound proteins, and ethanol-precipitated. Resuspended DNA was subsequently electrophoresed on a 1% agarose gel (1 × Tris acetate/EDTA) as described under ' Experimental Procedures. ' Lanes 1 in A and B contain 1-kb + DNA size markers (Invitrogen). B , stability of native complexes after digestion. Aliquots of 208-12 nucleosomal arrays were incubated with MeCP2 at the indicated r MeCP2 , digested with Eco RI at 37 ° Cfor 90 min, and electrophoresed as described for A . Arrowheads indicate the sizes of selected markers in base pairs.CC-BY · From the PDF
FIG. 6 Reconstructed image of an oligomer of MeCP2-bound nucleosomal arrays. Shown is a 0 ° projection of the tomographic reconstruction of an oligomer observed in negative stain. The oligomer ( inset ) was present in a sample assembled at r MeCP2 = 1.0. The interdigitating volumes of the nine 208-12 arrays comprising this oligomer are shown in a solid model, with each array numbered and represented in different colors. Bar = 20 nm.
FIG. 6 Reconstructed image of an oligomer of MeCP2-bound nucleosomal arrays. Shown is a 0 ° projection of the tomographic reconstruction of an oligomer observed in negative stain. The oligomer ( inset ) was present in a sample assembled at r MeCP2 = 1.0. The interdigitating volumes of the nine 208-12 arrays comprising this oligomer are shown in a solid model, with each array numbered and represented in different colors. Bar = 20 nm.CC-BY · From the PDF
Image 7
CC-BY · From the PDF

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  1. Lee, 2001, Brain Dev., 23, S138

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Metadata

Authors
Georgel, P. T., Horowitz-Scherer, R. A., Adkins, N., Woodcock, C. L., Wade, P. A., & Hansen, J. C.
Affiliations
  • The University of Texas at San Antonio Health Science Center (US)
  • University of Massachusetts Amherst (US)
  • Marshall University (US)
  • Emory University (US)
Corresponding author
  • Wade, Paul A.
  • Hansen, Jeffrey C.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2003-08
Volume
278
Issue
34
Pages
32181-32188
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m305308200
OpenAlex
W2042269850
Cited by
305
References
39
Citations by year
2026: 2 · 2025: 7 · 2024: 5 · 2023: 6 · 2022: 11 · 2021: 11 · 2020: 7 · 2019: 7 · 2018: 10 · 2017: 14 · 2016: 16 · 2015: 18 · 2014: 14 · 2013: 20 · 2012: 14
FWCI
5.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Collection of Biostatistics Research Archive (COBRA) (submittedVersion)
Funders
  • National Institutes of Health (5k22hd001238-02, 5r01gm043786-11)
  • National Institute of General Medical Sciences (GM 45916, GM43786)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (5K22HD01238)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
Keywords
MeCP2, chromatin condensation, nucleosome arrays, chromatin silencing, transcriptional repression, chromatin organization
MeSH terms
Chromatin, Chromosomal Proteins, Non-Histone, DNA-Binding Proteins, Humans, Recombinant Proteins, Repressor Proteins, Protein Structure, Secondary, DNA Methylation, Methyl-CpG-Binding Protein 2

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