RUNX1A affects gene regulation by displacing endogenous RUNX1C. (A) Experimental setup of CUT&RUN on murine Gata1s-FLCs after doxycycline-induced expression of HA-RUNX1A/RUNX1C. CUT&RUN was performed using anti-HA, anti-RUNX1(C-term), and anti-GATA1 antibodies. Peaks were called by SEACR47 using EV control cells as background. Data represent 1 experiment. (B) Venn diagram showing the number of genomic regions bound by endogenous RUNX1, endogenous GATA1s, and/or HA-RUNX1A after HA-RUNX1A overexpression. (C) Heat maps depicting the colocalization of endogenous GATA1s (orange, left), endogenous RUNX1 (green, middle), and HA-RUNX1A/-RUNX1C (red, right) signals after doxycycline-induced HA-RUNX1A or HA-RUNX1C expression in Gata1s-FLCs. EV-transduced cells were used as a control. Regions ±2.5 kilobases (kb) of the peak center are shown. Binding intensities are represented as normalized reads per kilobase of transcript per million reads mapped values (bars below the blots). (D) Binding intensities (normalized read density) of RUNX1 (left) and GATA1s (right), in EV control and doxycycline-induced HA-RUNX1A and HA-RUNX1C expressing cells. (E) Transcription factor motif enrichment analysis under RUNX1 peaks, at the promoter regions of genes that are differentially expressed upon doxycycline-induced RUNX1A expression (log2 fold change = 1). Human promoter regions were used as background. z score intensities are shown. (F) Integrative Genomics Viewer snapshots of Mxd1 and Lyl1 gene promoters showing occupancy of endogenous GATA1s, RUNX1, and HA-RUNX1A/-RUNX1C in Gata1s-FLCs after doxycycline-induced HA-RUNX1A/HA-RUNX1C expression or in EV control–expressing cells. The tracks display coverage (reads per kilobase of transcript per million reads mapped) (left). Scale and chromosome location are shown (top).
