Fig. 2: Point mutations in GATA2 C-terminal zinc finger associated with leukaemia and Emberger syndrome reduce mitotic retention.
From: GATA2 mitotic bookmarking is required for definitive haematopoiesis
a Representation of GATA2 domains highlighting leukaemia and Emberger syndrome (ES)-associated point mutations in the N- and C- terminal zinc fingers (ZF) of DNA-binding domain (DBD). TAD – transactivation domain. NRD – negative regulatory domain. NLS – nuclear localisation signal. CML – chronic myeloid leukaemia. AML – acute myeloid leukaemia. MDS – myelodysplastic syndrome. b Live-cell images of 293T cells overexpressing mCherry (mCh)-GATA2 deletion (Δ) constructs (red) excluding the N-terminal (amino acids 1-235), N-ZF (287–342), C-ZF (243–379) or NLS (380–440) in interphase (Inter) and metaphase (Meta). Mitotic events: n(ΔN-Terminal) = 395, n(ΔN-ZF) = 375, n(ΔC-ZF) = 100, n(ΔNLS) = 52. Scale bar, 10 µm. c, Protein expression in whole-cell extracts (WCE), cytoplasmic (Cy), soluble nucleus (SN) and chromatin-bound (Chr) fractions of both asynchronous (A, Async) and mitotic (M, Mit) 293T cells overexpressing deletion constructs. Representative blots were acquired with the same exposure time for asynchronous and mitotic cells. kDA, kilodaltons. d, Western blot quantifications of GATA2 deletions at Chr fraction in asynchronous and mitotic cells after normalising to H3. n(GATA2) = 5, n(ΔN-Term) = 4, n(ΔN-ZF) = 3, n(ΔC-ZF) = 4, n(ΔNLS) = 3. Statistical analysis performed by two-way ANOVA followed by Fisher’s LSD test and comparisons of mitotic retention of each deletion to GATA2 are shown. a, p = 0.005; b, p = 0.89, c and d, p = 0.01. e Protein expression of mCherry-fused GATA2 mutants in the Cy, SN and Chr protein fractions of asynchronous and mitotic 293T. Representative cells in metaphase are shown (right). Histone 2B (H2B)-mTurquoise signal (blue) indicates DNA content. Scale bar, 10 µm. f Quantification of GATA2 mutants at Chr fraction. n(GATA2) = 3, n(L359V) = 4, n(R362Q) = 4, n(R396Q) = 4, n(R398W) = 4, n(T354M) = 3, n(R361L) = 4, n(C373R) = 3. Statistical analysis performed by two-way ANOVA followed by Fisher’s LSD test and comparisons of mitotic retention of each mutant to GATA2 are shown. a, p = 0.86; b, p = 0.17, c and f, p = 0.01; d, p = 0.03; e and g, p = 0.02. g Hemogenic reprogramming efficiency (% CD9+CD49f+ cells) with GATA2, L359V or C373R, plus GFI1B and FOS. M2rtTA (M2) was used as control. n(M2) = 10, n(GATA2) = 13, n(L359V) = 11, n(C373R) = 11. Mean ± SD is represented. Statistical significance was analysed by one-way ANOVA followed by Bonferroni’s test. a and b, p < 0.001. Source data are provided as Source Data file.
