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. Author manuscript; available in PMC: 2022 Nov 30.
Published in final edited form as: Nat Cell Biol. 2022 Feb 24;24(3):384–399. doi: 10.1038/s41556-022-00850-x

Fig. 1|. EZH2 exhibits noncanonical PRC2-indepedent solo-binding in leukaemias.

Fig. 1|

Besides its canonical H3K27me3 cobound pattern, EZH2 exhibits additional noncanonical solo-binding at sites enriched for gene-activation-related histone marks, Pol II, (co)activators and cMyc in MLL-r leukaemia cells. a-d, Averaged signal intensities (a-b) and heatmaps (c-d) for EZH2 (either endogenous EZH2 or exogenously expressed HA-tagged EZH2), H3K27me3, histone acetylation (H3K27ac or H3K9ac) and H3K4me3 ± 5 kb from the centers of noncanonical EZH2+/H3K27me3- peaks (i.e., EZH2-”solo”; top panels) or canonical EZH2+/H3K27me3+ peaks (i.e., EZH2 ensemble; bottom panels) in either MV4;11 (a, c) or EOL-1 (b, d) cells. Except HA-EZH2, which was mapped via CUT&RUN, all the others were mapped via ChIP-seq.

e, Venn diagram showing a significant overlap between EZH2-”solo”-binding sites identified in the two independent MLL-r leukaemia lines, MV4;11 and EOL-1.

f-i, Heatmap for ChIP-seq signals of EZH2, POL II (f), MLL (MLLn and MLLc, g), BRD4 (h) and SWI/SNF (SMARCA4 and SMARCC1, i) ± 5 kb from the centers of EZH2-”solo”-binding sites in MV4;11 or EOL-1 cells.

j, Significant enrichment of the Myc:MAX motif (CACGTG) at the EZH2-”solo” peaks in MV4;11 cells.

k-l, Co-IP for EZH2 (k) or cMyc (l) interaction with endogenous POL II, SMARCA4 and SMARCB1 in EOL-1 cells using anti-EZH2 or anti-cMyc antibodies.

m-n, Co-IP for endogenous EZH2 and cMyc interaction in EOL-1 cells using anti-EZH2 (m) or anti-cMyc antibodies (n). Classic PRC2 subunits, SUZ12 and EED, and MAX, the cMyc cofactor, were probed in IP samples.

o-p, GST pulldown for assaying interaction of the in vitro translated HA-cMyc protein with recombinant GST-EZH2 (o) or GST-EED (p) protein. GST alone (lane 1) serves as control. Red arrows indicate GST and GST-fusion protein.

q-r, Averaged ChIP-seq signal intensities (q) and heatmaps (r) for EZH2 and cMyc (using two independent antibodies, ab1 and ab2) ± 5 kb from the centers of noncanonical EZH2-”solo” peaks (left) or canonical EZH2 ensemble peaks (right) in MV4;11 (top) or EOL-1 (bottom) cells.

s, Heatmaps for EZH2, H3K27me3, cMyc and H3K27ac signal intensities, detected by CUT&RUN in the MLL-AF9+ AML PDX cells, ± 5 kb from the centers of either EZH2-”solo” (top) or EZH2 ensemble (bottom) sites identified in MLL-AF4+ MV4;11 cells.