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. Author manuscript; available in PMC: 2024 Apr 12.
Published in final edited form as: Sci Transl Med. 2023 Sep 20;15(714):eadi7244. doi: 10.1126/scitranslmed.adi7244

Fig. 5. TBL1XR1::TP63 activates enhancers to confer PRC2 dependence.

Fig. 5.

(A) Ranked dependency score of genome-wide CRISPR screen in SMZ1 cells with TP63 and EZH2 highlighted. (B, C) Z scores for TP63 (B) and EZH2 (C) dependency across a pan-cancer set of 391 cell lines (14,30). (D) Cell proliferation assay of SMZ1 cells transduced with shRNAs targeting EZH2 using CTG. (E) IC50 of Valemetostat (VAL) for TCL cell lines. The B-cell lymphoma cell line Karpas-422 (blue), which harbors an EZH2 Y646F gain-of-function mutation serves as a positive control. TCL cell lines with TP63 rearrangements are in red and those with WT TP63 are in black. (F) Cell proliferation assay of SMZ1, DL40, and OCI-Ly12 cell lines treated with VAL using CTG. (G) Venn diagram showing genes regulated by TBL1XR1::TP63 from RNA-seq and genes bound by TBL1XR1::TP63 from ChIP-seq in SMZ1. (H) Possible mechanisms of regulating EED and EZH2 by TBL1XR1::TP63. (I, J) RT-qPCR analysis for EED, MYC, and EZH2 after TP63 knockdown with Dox treatment in SMZ1 cells (I) and MTA cells (J). *** P<0.001 or ****P<0.0001 for shTP63+Dox compared to shSeed+Dox for the same gene. (K, L) Western blot of EED, MYC, EZH2 and H3K27me3 after Dox-induced knockdown in Cas9-SMZ1-sgwtTP63 cells (K) and MTA cells (L). (M) The TBL1XR1-TP63-activated gene MYC is connected to a high-confidence distal TBL1XR1::TP63 binding site through chromatin loops identified from H3K27Ac HiChIP assays in Cas9-SMZ1-sgwtTP63 cells. HiChIP anchors that are looped to promoters of the target genes are presented. ChIP-seq shows the TBL1XR1::TP63 binding site as well as enhancer-associated chromatin marks. (N) ChIP-qPCR of TP63 fusion at the MYC enhancer in Cas9-SMZ1-sgwtTP63 cells. Control (CTR) primer sets are also included. (O) Luciferase reporter assay measuring the MYC enhancer activity in 293T cells expressing indicated proteins. The pGL3 plasmid without the MYC enhancer region (Empty vector, EV) is used as a negative control. (Y-axis) Relative (Rel.) Luciferase units are normalized to Renilla signal ± SD. (P) RT-qPCR analysis of MYC expression in SMZ1 cells with (+DOX) and without (−DOX) KRAB-dCas9 mediated repression of the MYC enhancer. (Q) PRO-seq reads at the MYC locus in Cas9-SMZ1-sgwtTP63 cells with (+DOX) and without (−DOX) TBL1XR1::TP63 knockdown using Dox-inducible shRNA.

Data are presented as mean ± SD, n=3 biological replicates. *** P<0.001 or ****P<0.0001 as compared between indicated groups (Student’s t-test).

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