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Fig. 2. RSF1 binds H2Aub nucleosomes through a previously uncharacterized UAB domain. (A) Schematic representation of the RSF1 domain organization and fragments used in nucleosome pull-down assay. (B) The full-length and fragment 7 (amino acids 770–807) of RSF1 bind to H2Aub nucleosomes. (Top) CBB-stained protein gel containing purified full-length RSF1 and its serial fragments. F1: 1–97 aa; F2, 98–182 aa; F3, 183–397 aa; F4, 398–608 aa; F5, 609–690 aa; F6, 691–785 aa; F7, 770–807 aa; F8, 808–890 aa; F9, 891–941 aa; F10, 942–1,441 aa. (Middle) Pull-down assays show that only the full-length RSF1 and fragment 7 (designated as the UAB domain) bind to H2Aub nucleosomes. (Bottom) Treatment with the H2Aub-specific deubiquitinase USP16 abolishes binding of RSF1 and fragment 7 to the nucleosomes. (C) The UAB domain specifically pulls down reconstituted nucleosomes subjected to in vitro ubiquitination by PRC1 (lane 2), but not unmodified nucleosomes (lane 1). (D) The UAB domain specifically pulls down H2Aub (lane 1), but not H2Bub (lane 2) nucleosomes. (E) The UAB domain is required for RSF1 to interact with H2Aub nucleosomes in 293T cells. Cells were transfected with an empty vector or vectors expressing Flag-RSF1 or mutated Flag-RSF1 (MT) in which the UAB domain was in-frame deleted. Mononucleosomes were prepared and subjected to anti-Flag IP followed by immunoblots. (F) Metaplot of Flag-RSF1 in control (blue line) and Ring1B KO (red line) mouse ESCs. For all data, the minimum value is set to 0. The signal plot is normalized by subtracting IgG ChIP-seq signal from the same cell lines. Transcription start site (TSS) plus 10-kb upstream and transcription termination site (TTS) plus 10-kb downstream are shown. (G) Venn diagram shows the overlap between RSF1 bound genes and H2Aub marked genes in mouse ESCs. About 82% of H2Aub sites are bound by RSF1. Image published in: Jones AE et al. (2017) Copyright © 2017. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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