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. 2021 Aug 25;10:e65872. doi: 10.7554/eLife.65872

Figure 1. Trithorax homology domain 2 (THD2)-mediated functions promote MLL-fusion-dependent leukemic transformation.

(A) Structure/function analysis of MLL-ELL. Various MLL-ELL constructs were examined for the transformation of myeloid progenitors. HA-tag (h: indicated as a red triangle) was fused to MTM and MTMT constructs. A schema of myeloid progenitor transformation assay is shown on top. Hoxa9 expression normalized to Gapdh in first-round and second-round colonies (left) is shown as the relative value of MLL-ELL (arbitrarily set at 100%) with error bars (mean ± SD of PCR triplicates). Colony-forming ability at third- and fourth-round passages (right) is shown with error bars (mean ± SD of ≥3 biological replicates). IBD: integrase-binding domain; hMBM: high-affinity MENIN-binding motif; LBD: LEDGF-binding domain; THD1/2: trithorax homology domains 1 and 2; SET: Suver3-9/enhancer-of-zeste/trithorax motif; TED: transcription elongation domain; OHD: occludin homology domain. (B) Requirement of THD2 for various MLL fusion proteins in leukemic transformation. Various MLL fusion constructs were examined for the transformation of myeloid progenitors as in (A). RA1/2: RAS association domains 1/2; PDZ: PSD-95/Dlg/ZO-1 domain; OM: octapeptide motif; LZ: leucine zipper. MISD: minimum interaction site of DOT1L: YEATS: Yaf9 ENL AF9 Taf14 Sas5 domain; AHD: ANC1 homology domain. (C) Colony morphologies of MTMTh or MTMh-ENL′ transformed cells. The colonies on day 5 of fourth passage are shown with a scale bar. The ratio of compact colonies (≥100 total colonies were counted in each experiment) is shown on the left (mean ± SD of six biological replicates). Representative images are shown on the right.

Figure 1.

Figure 1—figure supplement 1. Trithorax homology domain 2 (THD2) promotes MLL fusion-mediated leukemogenesis.

Figure 1—figure supplement 1.

(A) Protein expression from various MLL fusion constructs. Virus-packaging cells transiently expressing each MLL fusion construct shown in Figure 1A and B and (B) were analyzed by western blotting with the indicated antibodies. The expected bands corresponding to the transgene products are indicated by a red arrow head. (B) Negative effects imposed by the loss of THD2 domain on transformation ex vivo. Various MLL fusion constructs lacking the THD2 domains were examined for the transformation of myeloid progenitors as in Figure 1A. (C) Negative effects imposed by the loss of THD2 domain on transformation in vivo. Leukemogenic activity of MLL fusion constructs was examined. Each MLL fusion construct was transduced to c-Kit-positive hematopoietic progenitors and transplanted into syngeneic mice (n=10). Statistical analysis was performed using the log-rank test and Bonferroni correction with the wild-type control. *p ≤ 0.05, ****p ≤ 0.0001.