GATA2AS is localized to genomic sites enriched for erythroid transcription factors. (A) Genomic distribution of GATA2AS binding sites in uninduced HUDEP2 cells (day 0). GATA2AS peaks were separately called from odd and even probes, and 6793 overlapped peaks were used. (B) Distances to a TSS of GATA2AS binding sites were plotted by GREAT. (C) Known motifs enriched in GATA2AS binding sites were analyzed with HOMER and representative motifs of erythroid transcription factors are shown. q-values for all motifs are <0.0001. (D) Biotin-labeled GATA2AS RNA was used to pulldown GATA2AS-interacting proteins. Western blotting was used to detect LRF, KLF1, GATA1, and TAL1. (E) Interactions between GATA2AS and LRF or KLF1 were detected by RIP. RNAs interacting with LRF or KLF1 were detected by RT-qPCR. Normal IgG antibody and GAPDH RNA were used as controls. (F) Heat maps showing GATA2AS peaks grouped into 2 sets depending on overlap with LRF peaks. Published ChIP-seq data of LRF, GATA1, KLF1, TAL1, BCL11A, and LDB1 in HUDEP2 cells33,34 were plotted on the 2 sets of GATA2AS peaks. (G) Venn diagram showing GATA2AS-associated regions by GREAT and their overlap with GATA2AS DEGs on days 0-9 of differentiation. (H) GO term analysis of 1551 of GATA2AS associated genes by GREAT overlapped with GATA2AS KO DEGs (supplemental Figure 3C). IgG, immunoglobulin G.
