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. 2020 Feb 17;48(7):3542–3552. doi: 10.1093/nar/gkaa102

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — MeCP2 [1–205] binds to non-methylated GT-rich DNA in vitro and requires a methyl group provided by thymine. (A) Coomassie stained gel of purified recombinant proteins used in this study, full-length MeCP2[1–486], MeCP2[1–205] and MeCP2[77–167]. M = protein standard (Precision Plus, Bio-Rad). (B) Schematic diagram of the fragments of MeCP2 used here and in earlier studies. The lower diagrams show fragments of chicken MeCP2 tested previously for binding to GT-rich sequences (MAR) with high affinity, indicating whether GT binding was detected (20). The human MBD (grey shading) is located between amino-acids 78 and 162 (14). The highly homologous chicken MBD (also grey) extends from amino-acid 79 to 163. (C) EMSAs using varying amounts of MeCP2[1–205] or no protein (−) with probes containing non-methylated CG (CG), methylated CG (mCG), methylated CAC (mCAC) or GGTGT. (D) Graph showing quantification of MeCP2[1–205] binding to probes containing CG (filled squares), mCG (filled circles), mCAC (filled triangles) or GGTGT (crosses). Mean percentage of probe shifted (±SEM) based on triplicate experiments. (E) EMSAs using no protein (−) or varying amounts of MeCP2[1–205] with probes containing GGTGT, GGUGU, GGUGT or GGTGU to assess the influence of the thymine methyl group on binding.