Biochemical, biophysical and structural study of the nucleosome-MeCP2 complex
| dc.contributor.author | Yang, Chenghua, author | |
| dc.contributor.author | Luger, Karolin, advisor | |
| dc.date.accessioned | 2024-03-13T20:28:05Z | |
| dc.date.available | 2024-03-13T20:28:05Z | |
| dc.date.issued | 2009 | |
| dc.description.abstract | Methyl-CpG Binding Protein (MeCP2) is an abundant chromatin associated protein that is important in maintaining human health; mutations in this protein cause Rett Syndrome, a neurodevelopmental disease that is a common cause of mental retardation and autism in females. MeCP2 was initially identified as a protein that recognizes the genetic DNA methyl-CpG mark and it was thought to repress gene transcription by recruiting histone deacetylases. Recent studies show that MeCP2 can both repress and activate gene transcription. It also binds chromatin in the absence of the methylation mark, suggesting that its mode of action is more complex than previously assumed. The observation that MeCP2 compacts nucleosomal arrays in vitro and mediates silent chromatin loop formation in vivo suggests a novel mechanism by which MeCP2 regulates gene expression. To further characterize the interplay between MeCP2 and chromatin, it is important to understand the interactions between MeCP2 and nucleosomes, the fundamental component of chromatin. We used biochemical and biophysical approaches to study the interplay between MeCP2 and nucleosomes. Gel mobility assays showed that although MeCP2 can interact with a nucleosome with or without extra nucleosomal DNA, it has a higher affinity for nucleosomes with extra nucleosomal DNA. The N-terminal portion of human MeCP2 (amino acids 78-305) is sufficient to establish this interaction. Size-exclusion chromatography combined with multi-angle light scattering and fluoresecence resonance energy transfer (FRET) assays demonstrated that this interaction occurs at a 1:1 molar ratio and that MeCP2 brings the extra nucleosomal DNA ends in a closer proximity. Small angle X-ray scattering (SAXS) revealed the formation of a more compact complex when MeCP2 interacts with nucleosome with (versus without) extra nucleosomal DNA, indicating that the extra nucleosomal DNA is important in organizing the MeCP2-nucleosome complex. Our data suggest a model in which MeCP2 compacts chromatin by changing the extra nucleosomal DNA path. X-ray crystallography is also used to characterize the nucleosome-MeCP2 complex. Crystals of the nucleosomes with extra nucleosomal DNA in complex with MeCP2 were obtained and diffracted to 5.2 Å. Although MeCP2 dissociated from the crystals after soaking in cryo-protectant, the electron density map reveals the path of extra nucleosomal DNA which may be organized by MeCP2. | |
| dc.format.medium | born digital | |
| dc.format.medium | doctoral dissertations | |
| dc.identifier | ETDF_Yang_2009_3385142.pdf | |
| dc.identifier.uri | https://hdl.handle.net/10217/238032 | |
| dc.language | English | |
| dc.language.iso | eng | |
| dc.publisher | Colorado State University. Libraries | |
| dc.relation.ispartof | 2000-2019 | |
| dc.rights | Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright. | |
| dc.rights.license | Per the terms of a contractual agreement, all use of this item is limited to the non-commercial use of Colorado State University and its authorized users. | |
| dc.subject | methyl-CpG binding protein | |
| dc.subject | nucleosomes | |
| dc.subject | Rett Syndrome | |
| dc.subject | biochemistry | |
| dc.title | Biochemical, biophysical and structural study of the nucleosome-MeCP2 complex | |
| dc.type | Text | |
| dcterms.rights.dpla | This Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). | |
| thesis.degree.discipline | Biochemistry and Molecular Biology | |
| thesis.degree.grantor | Colorado State University | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) |
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