Cryo-electron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination

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Fig. 1 A Agarose gel shows DNA of native chromatin isolated from K562 cells and fractionated by ultracentrifugation of sucrose gradients (lanes 1-13), DNA size markers (lane 14 (M)), and total soluble chromatin (lane S2). Fig. 1 C DNP agarose gel showing native electrophoresis of the K562 chromatin crosslinked by glutaraldehyde at the indicated concentration of Mg2+ (lanes 2 – 8). Lane 1 (M): DNA size markers. Fig. 1 D (0 mM) TEM of the K562 chromatin crosslinked by glutaraldehyde at 0 mM Mg2+ Fig. 1 D (0.75 mM) TEM of the K562 chromatin crosslinked by glutaraldehyde at 0.75 mM Mg2+ Fig. 1 D (1.25 mM) TEM of the K562 chromatin crosslinked by glutaraldehyde at 1.25 mM Mg2+. Fig. 1 D (2.0 mM) TEM of the K562 chromatin crosslinked by glutaraldehyde at 2.0 mM Mg2+. Fig. 5 A (1-4) 18% SDS-PAGE gel stained by Coomassie R250 shows histones of control (lanes 1) and PAD4-treated (lanes 2, 3) nucleosome arrays from K562 cells. Lane 4: molecular mass markers. Fig. 5 A (5-7) SDS-PAGE and Western blotting of histones from control (lane 5) and PAD4-treated (lanes 2, 3) nucleosome arrays detected by antibodies against histone H3 citrullinated at Arginine 2, 8, and 17 Fig. 5 A (8-9) Triton-Acetate-Urea gel stained by Coomassie R250 shows histones extracted from control (lane 8) and PAD4-treated (lanes 9) nucleosome arrays from K562 cells. Fig. 5 B DNP agarose gel showing DNA size markers (lane 1) and native electrophoresis of the control (lanes 2-8) and PAD4-treated (lanes 9-15) nucleosome arrays from K562 cells. Fig. 6 A (1-5) 18% SDS-PAGE gel stained by Coomassie R250 shows histones of control (lanes 1, 3) and PAD4-treated (lanes 2, 4) reconstituted nucleosome core arrays. M – m.w. markers (lane 5). Fig. 6 A (6-10) Lanes 6-10: 18% SDS-PAGE gel stained by Coomassie R250 shows histones of control (lanes 7, 9) and PAD4-treated (lanes 8, 10) reconstituted nucleosome arrays additionally reconstituted with linker histone H1o after PAD4 treatment. Fig. 6 B (1-3) DNP agarose gels showing DNA size markers (lane 1) and native electrophoresis of the control (lane 2) and PAD4-treated (lanes 3) reconstituted nucleosome core arrays crosslinked with glutaraldehyde at 1 mM Mg2+ Fig. 6 B (4-10) DNP agarose gels showing DNA size markers (lane 4) and control (lanes 5, 7, 9) and PAD4-treated (lanes 6, 8, 10) reconstituted 183x12+H1 arrays after PAD4 treatment crosslinked at 0 to 1 mM Mg2+ as indicated. Supp. Fig. S1 A 18% SDS-PAGE gel stained by Coomassie R250 shows molecular mass markers (1) and histones of K562 fraction #10 chromatin incubated at the indicated concentration of Mg2+ together with 10 nm fiduciary gold particles for 48 hrs. at +4oC (2 – 9). Supp. Fig. S3 A Lanes 1-6 (S3 A) correspond to lanes 5-11: DNA size markers (lanes 5, 7, 9, 11), DNA of native MNase-digested chromatin isolated from K562 cells (6), HeLa cells (8) and PN21 mouse retina cells (10).

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