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. 2018 Oct 16;46(21):11129–11143. doi: 10.1093/nar/gky920

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© The Author(s) 2018. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — Requirements of Spp1 domains for H3K4 methylation activity of the C762 Set1 complex. (A) A schematic diagram of full-length (FL) Set1 and C762 Set1 fragment along with associating subunits deduced from previous study (15). RRM (RNA recognition motif), RRM2 (RNA recognition motif 2), n-SET, SET, and post-SET (hatched box) domains within Set1 protein are depicted. H3K4 methylation activities of purified Set1Cs on free histone H3 and recombinant chromatin templates assembled with unmodified H2B or H2Bub-containing octamers (Supplementary Figure S1A and B) are summarized on the right. (B) A schematic diagram of the C762-based Set1 complexes containing wild-type (WT) or mutant Spp1. (C) Requirement of the PHD domain of Spp1 for binding to histone H3 peptides with mono- (H3K4me1), di- (H3K4me2), tri- (H3K4me3), or no (H3K4me0) methylation at lysine 4 residue. Biotin pull-down assays employed biotinylated H3 peptides and purified FLAG-Spp1 proteins (Supplementary Figure S1E) and peptide-bound proteins were scored by immunoblotting with anti-FLAG antibody. (D) Requirement of the SID domain of Spp1 for binding to the n-SET domain of Set1. GST pull-down assays employed GST-tagged n-SET fragment (Set1 residues 762–937) (Supplementary Figure S1F) and purified FLAG-Spp1 proteins (Supplementary Figure S1E) and bound proteins were scored by immunoblotting with anti-FLAG antibody. (E) SDS-PAGE/Coomassie blue staining and anti-Spp1 immunoblot analyses of purified C762-based Set1 complexes reconstituted with baculoviruses expressing FLAG-tagged C762 Set1, Swd1, Swd3, Bre2, Sdc1, and either WT or mutant Spp1. Spp1 polypeptides are marked by asterisks. (F and G) Requirement of the PHDL domain of Spp1 for in vitro H3K4 methylation activity of Set1C on H2Bub chromatin. Free histone H3 (F) and H2Bub chromatin (G) were subjected to in vitro histone methyltransferase (HMT) assays with indicated purified C762-based Set1 complexes (E). H3K4 methylation status was monitored by immunoblotting with indicated antibodies. (H) Requirement of the PHDL domain of Spp1 for H3K4 methylation in yeast cells. Plasmids expressing WT or mutant Spp1 were introduced into the set1Δspp1Δ yeast strain that contains a chromosomal gene expressing HA (three copies)-tagged C762 Set1. Yeast whole cell extracts were analyzed by immunoblotting with indicated antibodies. H2Bub, K120-ubiquitylated H2B; H3K4me1, K4-monomethylated H3; H3K4me2, K4-dimethylated H3; H3K4me3, K4-trimethylated H3.