Figure 2. Germline RUNX1 variants influence transcription factor activity, subcellular localization, and CBFβ interaction.

(A and B) Luciferase reporter gene assay (driven by the PU.1 promoter in HeLa cells) showed minimal effects on transcription factor activity by missense RUNX1 variants identified in B-ALL (A) and T-ALL (B). A previously reported damaging variant p.R204Q was included as the reference for luciferase assay (red arrow). (C) Design of the Jurkat landing-pad system to measure RUNX1 variant activity in T-ALL. RUNX1 (either WT or variant) was inserted at the AAVS locus. EGFP coding sequence was knocked at the 3′ end of GZMA, a RUNX1 target gene. RUNX1 transcription factor activity was determined by flow cytometry of GFP signal, which reflects RUNX1-driven GZMA transcription. (D) Flow cytometry analysis of Jurkat cells expressing different RUNX1 variants. Cells harboring dominant-negative, loss-of-function, and WT-like RUNX1 variants exhibited the lowest, moderate, and highest GFP signals, respectively. (E) GFP signals from Jurkat cells expressing each RUNX1 variant (relative to WT) are shown in a bar graph. Data represent mean ± SEM (n = 3). The difference of each variant relative to EV was evaluated using Dunnett’s test. p.R204Q was used as control. *P < 0.05; **P < 0.01; ***P < 0.001.