Characterization of the chromatin binding domains of MeCP2

Abstract

Most molecular investigations have focused on MeCP2 as a transcriptional repressor in the context of Rett syndrome; however, the function of native MeCP2 in vivo is not understood. Previously in our laboratory, we demonstrated the capacity of human wild type (wt) MeCP2 to bind to unmethylated 208–12 DNA as well as defined 208–12 nucleosomal arrays in vitro. Concentration-dependent formation of complexes between MeCP2 and the chromatin occurred and electron microscopy revealed these structures to be highly condensed oligomers of MeCP2-bound nucleosomal arrays. The locations of the MeCP2 domain(s) that contribute to DNA binding and chromatin condensation have yet to be been defined; therefore, we hypothesize that there are multiple DNA binding domains on the MeCP2 protein and that these domains act cooperatively to condense chromatin. A cloning strategy designed to keep the putative MeCP2 functional domains intact was used to generate MeCP2 fragments aa 1–305, 1–78, 74–172, and 198–305. Sedimentation velocity experiments were performed on the MeCP2 proteins to determine whether MeCP2 was capable of oligomerization in solution. The fragments were tested for their ability to condense nucleosomal arrays. Using analytical ultracentrifugation and agarose electromobility assays we report that the MeCP2 protein functions in vitro as a monomer and has multiple DNA binding sites that permit chromatin condensation.