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. 2016 Aug 24;7:12464. doi: 10.1038/ncomms12464

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Copyright © 2016, The Author(s)

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(a) Commassie blue staining gel showing recombinant UHRF1 fusion proteins. The recombinant GST-UHRF1 (aa 95–610) and its YP191/192AA mutant were expressed and purified from E.coli. The YP191/192AA mutation in human UHRF1 was equivalent to the YP187/188AA mutation in mouse Uhrf1. (b) Diagram illustrating the in vitro reconstituted nucleosome with histone octamers containing with (or without) H3K9me2/3-containing H3. The 200 bp 601 sequence with a biotin at one end was used for in vitro nucleosome assembly via salt dialysis. (c) Verification of the histone composition of in vitro reconstituted nucleosomes with different H3K9 methylation status by Commassie blue staining gel. (d) Verification of in vitro reconstituted nucleosomes with different H3K9 methylation status by gel mobility shift assay. Asterisk marks free DNA. (e) The reconstituted nucleosomes were tested for binding to immobilized GST-UHRF1 by pulldown assay. Note only the nucleosomes with H3K9me2 or H3K9me3 bound to GST-UHRF1 but not the control GST. (f) Gel mobility assay examining the nucleosomes derived from in vitro assembly with histone octamers containing H3 without N-terminal tail or different levels of H3K9 methylation. (g) The nucleosomes assembled in (f) were immobilized to streptavidin agarose beads and assayed for binding of GST-UHRF1. The Commassie staining gel at lower panel showed the compositions of core histones in corresponding in vitro assembled nucleosomes. (h) and (i) The pulldown was performed as in (g) except the GST-UHRF1 YP191/192AA mutant proteins and GST-UHRF1 (aa 1–407) without SRA domain were used, respectively.