To probe the tertiary structure and domain organization of native methyl CpG-binding protein 2 (MeCP2), the recombinant human e2 isoform was purified to homogeneity and characterized by analytical ultracentrifugation, CD, and protease digestion. The location of intrinsic disorder in the MeCP2 sequence was predicted using the FoldIndex algorithm. MeCP2 was found to be monomeric in low and high salt and over a nearly 1000-fold concentration range. CD indicated that the MeCP2 monomer was nearly 60% unstructured under conditions where it could preferentially recognize CpG dinucleotides and condense chromatin. Protease digestion experiments demonstrate that MeCP2 is composed of at least six structurally distinct domains, two of which correspond to the well characterized methyl DNA binding domain and transcriptional repression domain. These domains collectively are organized into a tertiary structure with coil-like hydrodynamic properties, reflecting the extensive disorder in the MeCP2 sequence. When expressed as individual fragments, the methyl DNA binding domain and transcriptional repression domain both could function as nonspecific DNA binding domains. The unusual structural features of MeCP2 provide a basis for understanding MeCP2 multifunctionality in vitro and in vivo. These studies also establish an experimental paradigm for characterizing the tertiary structures of other highly disordered proteins.Methyl CpG-binding protein 2 (MeCP2) 2 is a 53-kDa nuclear protein named for its methylated DNA-binding capacity (1, 2). Accordingly, MeCP2 can preferentially recognize methylated DNA and act as a methylation-dependent transcriptional repressor in vitro and in vivo (3, 4). MeCP2 is also involved in the maintenance of condensed chromosomal superstructures in vivo (5, 6) and in vitro (7,8) and regulates mRNA splicing in vivo (9). MeCP2 is able to interact with many different macromolecules and macromolecular complexes, including unmethylated and methylated DNA (10 -12), nucleosomes and chromatin (7,8,13), transcriptional co-repressors (14), a histone H3 methyltransferase (15) 207-310 (20). Despite the extensive interest in MeCP2 function, very little is known beyond the tertiary structure of the protein outside of the MBD, and even this domain is not well understood at the biochemical level. For example, fully one-half of the residues required to recognize a single methylated CpG are disordered in the NMR structure of the MBD (21-23). The importance of deciphering the structural basis of MeCP2 function is further underscored by its central role in the neurological disorder, Rett Syndrome, which is caused by a number of different nonsense, missense, and frameshift mutations scattered throughout the Mecp2 gene (24 -26).Intrinsically disordered proteins have one or more long regions that do not on their own fold into ␣-helices or -sheets/ turns (27-31). Structural genomics studies indicate that many eukaryotic proteins contain one or more intrinsically disordered regions, including a large number involved in genome reg...
The accessibility of eukaryotic DNA is dependent upon the hierarchical level of chromatin organization. These include (1) intra-nucleosome interactions, (2) inter-nucleosome interactions and (3) the influence of non-histone chromatin architectural proteins. There appears to be interplay between all these levels, in that one level can override another or that two or more can act in concert. In the first level, the stability of the nucleosome itself is dependent on the number and type of contacts between the core histones and the surrounding DNA, as well as protein-protein interactions within the core histone octamer. Core histone variants, post-translational modifications of the histones, and linker histones binding to the DNA all influence the organization and stability of the nucleosome. When nucleosomes are placed end-to-end in linear chromatin arrays, the second level of organization is revealed. The amino terminal tails of the histone proteins make contacts with adjacent and distant nucleosomes, both within the fiber and between different fibers. The third level of organization is imposed upon these 'intrinsic' constraints, and is due to the influence of chromatin binding proteins that alter the architecture of the underlying fiber. These chromatin architectural proteins can, in some cases, bypass intrinsic constraints and impart their own topological affects, resulting in truly unique, supra-molecular assemblages that undoubtedly influence the accessibility of the underlying DNA. In this review we will provide a brief summary of what has been learned about the intrinsic dynamics of chromatin fibers, and survey the biology and architectural affects of the handful of chromatin architectural proteins that have been identified and characterized. These proteins are likely only a small subset of the architectural proteins encoded within the eukaryotic genome. We hope that an increased understanding and appreciation of the contribution of these proteins to genome accessibility will hasten the identification and characterization of more of these important regulatory factors.
Mutations in methyl DNA binding protein 2 (MeCP2) cause the neurodevelopmental disorder Rett syndrome (RTT). The mechanism(s) by which the native MeCP2 protein operates in the cell are not well understood. Historically, MeCP2 has been characterized as a proximal gene silencer with 2 functional domains: a methyl DNA binding domain and a transcription repression domain. However, several lines of new data indicate that MeCP2 structure and function relationships are more complex. In this review, we first discuss recent studies that have advanced understanding of the basic structural biochemistry of MeCP2. This is followed by an analysis of cell-based experiments suggesting MeCP2 is a regulator, rather than a strict silencer, of transcription. The new data establish MeCP2 as a multifunctional nuclear protein, with potentially important roles in chromatin architecture, regulation of RNA splicing, and active transcription. We conclude by discussing clinical correlations between domain-specific mutations and RTT pathology to stress that all structural domains of MeCP2 are required to properly mediate cellular function of the intact protein.
Bisphenol A (BPA) is a versatile petrochemical used in the preparation of high volume polymers including polycarbonates and epoxy resins. Unfortunately, BPA is also an endocrine disrupter and has been...
Amorphous copolyesters based on combinations of ethylene glycol (EG) and 1,4-cyclohexanedimethanol (CHDM) as diols with terephthalic acid have excellent properties, such as toughness, clarity, and chemical resistance and are well known commercial polymers. Several different hydoxyethoxylated bisphenols were copolymerized into the backbone of these amorphous copolyesters in order to raise the glass transition temperature (Tg). The incorporation of bis[4-(2-hydroxyethoxy)phenyl]sulfone (SEO-2) into the amorphous copolyester backbone led to significant Tg increase and improvement in resistance to lipids. Melt viscosity and other mechanical and physical properties were unchanged by the addition of SEO-2.
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