Extended Data Fig. 1. CTSG, ELANE, and PRTN3-Mediated H3ΔN in Monocytes.
(a) H3ΔNThr22 enrichment in monocytes is not associated with cell death. Viability of the cells shown in Fig. 1a measured by cisplatin staining. Data represent mean ± S.E.M. (N = 20).(b) CD14+CD16- classical monocyte-specific H3ΔNThr22 enrichment. EpiTOF analysis of the indicated monocyte subsets. Y-axis, normalized H3ΔNThr22 level; center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Statistical significance is determined by two-tailed Welch’s t-test with P values depicted.
(c) Inverse relationship between class II MHC expression and H3ΔNThr22 in monocytes. Single-cell analysis of EpiTOF data as in (a). Each dot represents a single monocyte. X-axis, HLA-DR; y-axis, H3ΔNThr22 (top) or bulk H3 levels (bottom).
(d) Chromatin localization of H3ΔN in monocytic cells. Western blot analysis of THP-1 cells biochemically separated into cytoplasmic and insoluble chromatin fractions.
(e) H3ΔN in monocytes is not catalyzed by cathepsin L. Western blot analysis of WCE from THP-1 cells expressing CRISPR-Cas9 and two independent sgRNAs targeting CTSL. Control cells express CRISPR-Cas9 but lack sgRNA.
(f) H3ΔN in monocytes is not catalyzed by matrix metalloproteases (MMPs). Western blot analysis of WCE from U937 (top) or THP-1 (bottom) cells treated with MMP9-specific or broad-spectrum MMP inhibitors. Control, DMSO treated.
(g) H3ΔN in monocytes is not catalyzed by cysteine proteases. Western blot analysis of WCE from U937 (left) or THP-1 (right) cells cultured in the presence of a cell-permeable broad-spectrum cysteine protease inhibitor E-64d. Control, DMSO treated.
(h) Serine proteases generate H3ΔN in monocytic THP-1 cells. Western blot analysis of WCE from THP-1 cells treated with nonselective serine protease inhibitor AEBSF. Control, PBS treated.
(i) Chromatin localization of CTSG, ELANE, and PRTN3. Western blot analysis of the cytoplasmic and chromatin-enriched fractions purified from THP-1 cells.
(j) Release of chromatin-bound NSPs in high-salt solution. Chromatin pellet as in Fig. 1d is washed extensively with a buffer containing physiological ionic strength and is subsequently treated with a high-salt solution to solubilize chromatin proteins. Supernatant (left) and pellet (right) fractions are subject to immunoblotting analysis. HP1α serves as a chromatin protein control.
(k) Controlled proteolytic activities of CTSG, ELANE, and PRTN3 on nucleosomal H3 in vitro. Tandem mass spectrometry analysis of protease assays as in Fig. 1h using individual NSPs and recombinant nucleosomes. Primary cleavage sites accounting for greater than 20% of proteolytic products are labeled. CTSG (red); ELANE (green); PRTN3 (blue).
(l and m) Distinct histone modification profiles between FL- and truncated H3. Immunoblotting analysis of WCE from U937 cells (l) or primary monocytes (m) using the indicated antibodies. Three biological replicates are shown. These results are used for the quantitative analyses shown in Fig. 1i and 1j.