Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
ReviewThe MBD protein family—Reading an epigenetic mark?
Introduction
In the late 1980s and early 1990s, Bird and colleagues established reliable biochemical assays for the detection and eventual biochemical isolation from mammalian cells of proteins with the capacity to selectively recognize methylated DNA [1], [2]. The founding protein, termed MeCP2, was demonstrated to possess a sequence motif both necessary and sufficient for methylated DNA binding [3]. Approximately 10 years ago, Hendrich and Bird published a landmark paper enlarging the family of mammalian proteins containing the methyl-CpG-binding domain to its current size [4]. Subsequent work has established that this protein motif is conserved across the eukaryotic lineage [5]. The intervening decade has witnessed considerable interest in these proteins, with characterization of their biochemistry and genetics. Here we highlight recent findings that suggest an unanticipated level of complexity to the biology of MBD proteins.
The MBD sequence motif was defined by molecular analysis of the prototype MBD protein, MeCP2 [3]. This sequence motif was found to be both necessary and sufficient to direct specific interaction with methyl-CpG-containing DNA fragments. It consists of approximately 70 amino acids and represents the sole sequence feature found in common in all MBD family members (Fig. 1A). The single exception to this rule is the case of MBD2 and MBD3, which are nearly identical in amino acid sequence, have common gene structures and are believed to have arisen from an ancient duplication in the evolution of the vertebrate lineage [5]. The primary amino acid sequence of the motif is remarkably conserved across the different family members within mammals (Fig. 1B).
The MBD sequence motif corresponds very closely with a structural domain now characterized at atomic level resolution. Solution structures of the MBD from multiple different MBD proteins as well as from different species adopt very similar folds [6], [7], [8], forming a wedge-shaped structure. The front face of the wedge consists of beta strands (which vary in number depending on the protein), the other face of the wedge contains a short alpha helix. The NMR structure of the MBD domain from MBD1 in complex with a short methylated duplex oligonucleotide [9] demonstrated a conformational change consistent with an induced fit mechanism on interaction with DNA. This finding has been confirmed recently by solution of the crystal structure of MeCP2 in complex with methylated DNA [10]. A pair of arginine residues and an aspartic acid residue, all three being absolutely conserved across the MBD family, make direct contact with DNA bases (Fig. 1). Further contacts between the protein and DNA recognition site are mediated by several highly structured water molecules, including a hydrogen bond made by a highly conserved tyrosine hydroxyl group (Fig. 1). Finally, the MeCP2 MBD domain contains an unusual Asx-ST motif that makes contact with a DNA backbone phosphate placed in an atypical structure (for standard B form DNA) by a local AT run [10], a sequence feature found associated with high-affinity MeCP2-binding sites in vitro and in vivo [11].
Section snippets
MeCP2 as a chromatin component
In the late 1990s a seminal set of observations established a biochemical link between the MBD family and enzymes that modify chromatin components. MeCP2 [12], [13], MBD3 [14], [15], MBD2 [15], [16], and MBD1 [17] were linked by biochemical and/or molecular analysis to histone deacetylase enzymes. These studies led to the propagation of a model suggesting that members of the MBD family bind specifically to methylated DNA through the action of the MBD motif where they recruit enzymatic
MeCP2 as an architectural component of chromatin
Mutations in the human MeCP2 locus are known to be causal for the autism spectrum disorder Rett Syndrome [24], prompting the creation of animal models. Initial characterization of the potential impact of MECP2 deficiency on the transcriptome in these models failed to yield substantial insights into genetic targets [25]. These studies were performed with whole brain tissue, potentially masking specific alterations of a cell type specific nature. In fact, convincing evidence emerged that MeCP2
Association of MeCP2 with active genes
The emergence of the genomic era has provided a wealth of new research tools for studying the function of components of chromatin. One particularly valuable tool has been the application of chromatin immunoprecipitation as a tool to generate DNA probes for high-density microarrays that tile regions of mammalian genomes. Such experiments have the potential to map, at high resolution, the association of proteins with genomic chromatin in living cells [32].
The application of this ChIP-chip
Summary and future perspectives
How do we rationalize the seemingly disparate stories describing MeCP2 biology? The in vitro biochemical properties of the protein seem to indicate that MeCP2 is highly unusual. It binds methylated DNA, but prefers a local sequence context [11]. It is also a general DNA-binding protein and the difference in affinity for any DNA compared to methylated DNA is not huge [19], [21]. High-resolution imaging of the protein bound to DNA indicates the presence of multiple DNA interaction surfaces and a
Conflict of interest
None.
Acknowledgements
This research was supported (Project number 1Z01ES101965) by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. We thank the members of the Wade laboratory for useful discussions throughout the course of preparation of this manuscript. We apologize to our colleagues whose work could not be cited here due to space considerations.
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2020, Coordination Chemistry ReviewsCitation Excerpt :To conquer this problem, methyl-CpG-binding domain protein family attracts researcher's interests. There are at least five mammalian MBD proteins, namely MeCP2, MBD1, MBD2, and MBD3 for transcriptional repression, and MBD4 (also known as MED1) for mismatch repair as a thymine glycosylase [127,128]. Among them, MeCP2 and MBD1 have been widely applied for symmetric DNA methylation recognition with high affinity though 5mC is located inside of dsDNA structure, which greatly solved the low recognition efficiency of anti-5mC antibody [129–131].