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. 2024 Apr 23;14(8):1522–1546. doi: 10.1158/2159-8290.CD-23-0876

Figure 5.

Figure 5.

Mutant ENL forms condensates at key target genes, and disrupting condensate formation impairs its oncogenic activity in HSPCs. A, Schematic representation of the transduction of various FLAG-tagged ENL transgenes in LSK cells and subsequent experiments (shown in C–G). B, Schematic depiction of domain structure and features of the ENL mutant protein. C, Representative images of anti-FLAG IF staining in LSK cells expressing the indicated FLAG-ENL transgenes. WT, ENL-WT; T1, ENL-T1; T4, ENL-T4; T1(H116P), H116P mutation in the YEATS domain of ENL-T1; T1(AHD_mut), DRD538RFR mutations in the AHD domain of ENL-T1; T1(IDR1_del), deletion of IDR1 in ENL-T1; T1(IDR2_del), deletion of IDR2 in ENL-T1. D and E, Percentage of nuclei with or without FLAG-ENL puncta (D) and the number of FLAG-ENL puncta (E) in each nucleus in LSK cells. Error bars represent mean ± SEM. P values using unpaired, two-tailed Student t test. F, RT-qPCR analysis showing mRNA expression of Hoxa5/6/9, Meis1, Igf2bp3, and Rnf43 in LSK cells expressing indicated FLAG-ENL transgenes. Error bars represent mean ± SEM (n = 3). G, Quantification of colonies formed by LSK cells expressing indicated FLAG-ENL transgenes. Error bars represent mean ± SEM (n = 3). P value using unpaired, two-tailed Student t test. H, Average occupancies (top) and heatmap (bottom) representation of FLAG-ENL-bound peak regions that show increased occupancy by T1 compared with WT ENL in LSK cells expressing the indicated FLAG-ENL transgenes. The CUT&Tag signals are normalized by reads per million (RPM). See Supplementary Table S32. I and J, Average occupancies (top) and heatmap (bottom) showing H3K27ac (I) and p300 (J) at genomic regions that exhibit T1-induced increase in H3K27ac and FLAG-ENL in LSK cells expressing the indicated FLAG-ENL transgenes. The CUT&Tag or ChIP-seq signal are normalized by RPM. See Supplementary Table S33. K, Schematic workflow of LSK cells transplantation experiment. L, Flow cytometric quantification of FLAG-ENL transgenes expressing cells (CD45.2+CD45.1−) in the PB harvested at indicated time points from C57BL/6 recipient mice. Bars represent the median (WT, n = 5; T1, n = 6; T1(H116P), n = 5). P value using unpaired, two-tailed Student t test. M, Kaplan–Meier survival curves of C57BL/6 recipient mice that received LSK cells expressing the indicated FLAG-ENL transgenes. WT, n = 5; T1, n = 6; T1(H116P), n = 5. The gray line (WT) overlapped with the blue line [T1 (H116P)]. P value using log-rank test.