Fig. 5
SUMO2-PCNA enhances deposition of CAF1-dependent histone H3.1. a DNA supercoiling assays to detect nucleosome assembly. Reactions were initiated by mixing cytoplasmic extracts (6 µg each) from HEK293T cells transfected with a control vector, or WT, K164R (KR), or SUMO2-KR (S2-KR) PCNA with purified, non-assembled histones (left panel) or pre-assembled H3-H4 tetramer and H2A-H2B dimer (right panel) in a reaction containing relaxed circular DNA plasmids (200 ng, linear length: 3.0 kb) pre-treated with topoisomerase I (Topo I). After deproteination to stop the reaction, DNA molecules were precipitated and analyzed by agarose gel electrophoresis. Supercoiled DNA, relaxed DNA, and topoisomers are indicated. b DNA supercoiling assays to detect nucleosome assembly using the indicated amounts of control or CAF1-depleted cytoplasmic extracts from S2-KR-overexpressing HEK293T cells. c (top) Western blot analysis of expression of the indicated FLAG-PCNA complexes in whole cell extracts (WCE) of HEK293T cells co-transfected with the indicated HA-tagged histone H3 or vector alone. Tubulin was used as a loading control. (bottom) Western blot analysis of HA-H3 immunopurified from the indicated cells with an HA antibody. d, e ChIP analysis of the samples from (c) for HA-H3.1 and HA-3.3 occupancy at the FRA7K (d) and FRAXC (e) gene regions. Each average value ± standard deviation was calculated from triplicate qPCR reactions per one representative experiment. p values were calculated by t-test analysis for statistically significant differences. p values equal to or less than 0.05 are indicated with an asterisk (*).The result was reproduced in three independent assays. f Western blot analysis of CAF1, RNAPII, histone H3, and H3 tri-methyl lysine 9 (H3K9me3) in WCE of HEK293T cells with or without FLAG-CAF1 overexpression. g Western blot analysis of FLAG-PCNA, MCM7, histone H3, and H3 di-methyl K9 (H3K9me2) and H3K9me3 in WCE of HEK293T cells transfected with the indicated FLAG-PCNA constructs. h Micrococcal nuclease (MNase) digestion assay of nuclei isolated from HEK293T cells overexpressing the indicated FLAG–PCNA proteins. The digested samples were deproteinized, and the DNA purified and analyzed by agarose gel electrophoresis
