Biochemical and Biophysical Research Communications
Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA
Introduction
The MeCP2 protein is highly expressed in neuronal cells in the brain and it has been reported that female mice lacking one copy of the gene encoding this protein show symptoms similar to those of girls with Rett syndrome [1]. MeCP2 binds to methylated CpG sequences as well as non-CG methylated DNA [2], [3], [4], [5], [6] and it can interact with nuclear receptor co-repressor (NCoR/SMRT), which forms a complex with Sin3A [7]. Then, Sin3A recruits HDAC, which removes acetyl groups from histones, resulting in the chromatin compaction and transcriptional repression. In addition, higher-order chromatin compaction [8] and large-scale chromatin looping [9] have been reported by the unmethylated DNA binding of MeCP2.
MeCP2 protein comprises a methylated-DNA binding domain (MBD; residues 78–163), transcriptional-repression domain (TRD; residues 206–310), and C-terminal domains (CTD-α and CTD-β; residues 311–355 and 356–486, respectively) [10]. The TRD domain overlaps with the nuclear receptor co-repressor interaction domain (NID; residues 285–313; in X-ray structure, residues 298–307 are in contact with TBLR1 [11]) and basic regions including an AT-rich DNA binding domain, AT-hook 2 [4]. DNA binding domains of MeCP2 recognize a wide range of sequences. While the N-terminal MBD recognizes and binds to methylated CpG [12], three AT-hook DNA binding motifs are found in the central and C-terminal region of MeCP2. Among them, the AT-rich DNA binding properties of AT-hook 1 (residues 184–195) and 2 (residues 264–273) have been characterized [13]. In addition to these motifs, a cluster of basic amino acids (residues 270–340) has been recognized as DNA-binding region [5].
The DNA-binding properties of MeCP2 are closely related to its function as an epigenetic regulator through the modification of chromatin structure [6]. Several recent analyses showed that there are multiple DNA binding motifs outside MBD in MeCP2, and these DNA binding regions are suggested to perform cooperative binding to DNA in the process of MeCP2 association with chromatin [6]. Recently, besides the three AT-hooks, the basic cluster (270–340) in MeCP2 has been shown to have a DNA-binding function, which is disrupted by mutation at R306 [5]. In this cluster, residues from K304 to R309 are especially basic and this region contains the Rett-causing mutation point R306. Besides the interaction of the NID, including R306, with the NCoR/SMRT co-repressor complex [11], [14], this basic cluster is also known to bind DNA by chromatin immunoprecipitation experiments and electrophoretic mobility shift assay (EMSA) [5]. Intriguingly, it is reported MeCP2 requires AT-rich sequences adjacent to methyl-CpG for the DNA binding, and MeCP2 with the loss of AT-hook domain still binds to these DNA [15]. This result strongly suggests that there is another AT-rich DNA binding domain in MeCP2, which is responsible for the cooperative binding to chromatin DNA with MBD.
To elucidate the DNA-binding properties of this region, we prepared peptide fragments including the basic cluster, comprised of residues 295–313 with N-terminal serine as a cloning artifact (hereafter referred to as basic cluster peptide 20, BCP-20) and residues 302–310 (hereafter referred to as BCP-9), and performed binding studies with AT-rich DNAs, DNA-1 (5’-CGAATTAATTCG-3’) and DNA-2 (5’-GCTTAATATGG-3’), by NMR and ITC. We found that the basic cluster peptides from the MeCP2 showed direct contact with the minor groove of AT-rich DNA, with an affinity similar to that of the AT-hook peptides. The binding characteristics of the BCPs were analyzed and the bound structure was modeled with NMR-data based docking and molecular dynamics simulations. These results suggest a potential role of the basic cluster of MeCP2 in chromatin binding and compaction.
Section snippets
Materials and methods
Sample preparation and the detailed experimental methods are described in the supplementary information.
The basic cluster in the TRD domain of MeCP2 contains an AT-rich DNA-binding motif
MeCP2 has three AT-hook motif sequences in its central and C-terminal regions, and these AT-hook motifs can be aligned with the AT-hook motifs in the high mobility group protein HMGA1, which is a non-histone chromatin protein. After alignment, found another basic cluster between AT-hook 2 and 3 motifs that is distinct from these AT-hook motifs, including the residues K304 to R309 that show the most basic properties (Fig. S1). In this basic cluster, R306 is known to be frequently mutated in Rett
Acknowledgements
We thank Dr. Huda Y. Zoghbi at Baylor College of Medicine for discussions that helped shape this study and for reading the manuscript. This work was supported by the grants NRF-2013M3A6A4045160, 2014R1A4A1007304 (Y.H.J.), and NRF-2016K1A1A2912057 (J.J.S.) of the National Research Foundation funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea.
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Structural investigation of Rett-inducing MeCP2 mutations
2019, Genes and DiseasesCitation Excerpt :Along the 486 amino acid MeCP2 sequence, there are unambiguous signals confirming the role of the protein in the interaction with DNA. Indeed, AT-hook domains, critical for altering chromatin structure, have been delineated in MeCP2 transcription repressor domains, TRD,2–4 together with a methyl-CpG-binding domain, MBD. Trying to correlate structural aspects of amino acid changes arising from missense MecP2 mutations with RTT is not straightforward, as the protein belongs to the class of intrinsically disordered proteins, IDPs,5 see Fig. 1.