Fig. 3.

H3K4 methylation by Uhrf1 complex is mediated via a Setd1a. a Immunoprecipitation analysis of FLAG-tagged Uhrf1 protein. Uhrf1 was co-immunoprecipitated with Setd1a/COMPASS complex proteins isolated from mESCs. The data in c–g are represented as n = 3 experimental replicates (independent HMT assay from different cell cultures). Values are mean ± S.D. ^∗∗P < 0.01, by unpaired, two-tailed Student’s t-test analysis. b Mutants of Uhrf1 deleted of specific domain (ΔUbi, Ubiquitin domain; ΔTTD, TTD domain; ΔPHD, PHD domain; ΔSRA, SRA domain) were used for co-immunoprecipitation with Setd1a. c SET domain of SETD1A is necessary for interacting with Uhrf1. Series of SETD1A constructs with given deletions were used for Co-IP with Uhrf1. d Uhrf1 complex isolated form mESCs was used for assaying the histone H3K4me3 methyltransferase activity with chromatin as substrate. e Setd1a is a histone methyltransferase in complex with Uhrf1. Depletion of Setd1a reduced the H3K4 methyltransferase activity in Uhrf1 complex. f SRA domain is essential for a histone methyltransferase activity of Uhrf1 complex. In vitro HMT assay with each of Uhrf1 deletion mutant constructs was performed with H3 as substrate. H3K4me3 modifications were not detected with SRA domain deletion