Fig. 6: Chromatin factors enforce differentiation blockade in AML through corrupted transcription factor interactions.
From: In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis
a, Heatmaps showing ChIP–seq signal for cBAF (Smarcb1), MLL (Kmt2a) and MLL4 (Kmt2d) in leukemia (Npm1c and Flt3-ITD), in vivo myeloid progenitors (GMPs), in vivo monocytes and ex vivo-derived primary monocytes. n = 2 independent ChIP–seq experiments per factor. b, Motif enrichment analysis of cBAF, MLL and MLL4 binding patterns specific for leukemia (leukemic), common between leukemia and myeloid progenitors (common), and specific for normal myeloid cells (normal). c, Box plot showing the Stat5a, Runx1 and Runx2 binding signal (ChIP–seq) at the leukemic, common and normal loci defined in Fig. 6a (n = 2). The number of loci comprising the leukemic, common and normal categories are 2,019, 581 and 2,361 for Smarcb1; 2,427, 2,346 and 5,789 for Kmt2a; and 3,144, 810 and 4,129 for Kmt2d. d, Genome browser tracks showing the ChIP–seq signal for Smarcb1, Kmt2a, Kmt2d, Stat5a and Runx2 in leukemia, and ATAC-seq for control and chromatin factor-depleted leukemia cells. The chosen loci are leukemic-specific. n = 2 independent experiments. The green highlighted regions shown identify chromatin factor–transcription factor binding and altered accessibility on chromatin factor knockout. e, Box plots showing changes in chromatin accessibility at leukemic loci bound by Stat5a, Runx1 and Runx2 on depleting specific chromatin factors. cBAF, n = 2 independent experiments. Smarcd2 knockout: n = 1,385, 1,050, 1,711; statistic = 0.73, 0.75, 0.76; P = 5 × 10−15, 0, 0. Kmt2a knockout: n = 1,288, 695, 1,427; statistic = 0.78, 0.86, 0.78; P = 0, 9 × 10−16, 0. Kmt2d knockout: n = 1,482, 990, 1,913; statistic = 0.70, 0.70, 0.69; P = 2 × 10−15, 0, 0. The decay in accessibility was tested statistically using a two-sided Kolmogorov–Smirnov test. f, Growth curves for Cas9-leukemic cells expressing NTC and anti-Stat5a sgRNAs, n = 3 independent experiments. ***P < 0.001 (two-way ANOVA). The error bars are the s.e.m., the midpoints show the mean. The box plots in c and e display the median and the distribution’s 25th (minima) and 75th (maxima) percentiles. The whiskers extend up to 1.5 times the IQR (Q3–Q1) from the minima and maxima.
