Figure 3

Specific binding with native mononucleosome library by reader modules in matrix-assisted reader chromatin capture (MARCC). (A) Scheme of MARCC. Reader domain characterized by peptide microarray is immobilized on resin and incubated with MNase-digested native mononucleosomes. Enriched nucleosomes are released by TEV protease cleavage and analyzed for coexisting histone PTMs and associated DNA. See text for details. (B,C) AIRE-PHD, ING2-PHD and ATRX-ADD were immobilized on resin and incubated with native mononucleosomes. Bound nucleosomes were boiled on beads and separated on 12% SDS-PAGE and probed with corresponding histone antibodies. HaloTag protein was included as a negative control. Bound nucleosomes were quantified as percentage input. (D) ING2-PHD MARCC enriched for active chromatin. qPCR for active chromatin marker GAPDH and heterochromatin marker SAT-2 was performed for DNA extracted from MARCC by ING2-PHD and ChIP by H3K4me3 antibody (ab8580). IgG and HaloTag were used as negative controls for MARCC and ChIP, respectively. The DNA was quantified by input DNA standard curve and plotted as percentage input.