Figure 7. GFI1B is marked by H3K27me3 in Kasumi-1, but not CD34+ cells expressing AML1-ETO.
(A) RNA-seq revealed a significant increase in GFI1B expression following AML1-ETO degradation in CD34 cells, but not Kasumi-1 cells. (B and C) CUT&RUN analysis of H3K27me3 in Kasumi-1 and CD34+ HSPCs expressing AML1-ETO-FKBP-HA. (B) Venn diagram shows H3K27me3 marked genes that overlap between these cell types. (C) Heat maps of CUT&RUN peaks in Kasumi-1 (left) or CD34+ AML1-ETO expressing cells (right). (D and E) IGV screenshots of an AML1-ETO target gene unique to CD34+ cells (GFI1B; D) and an AML1-ETO target gene shared between Kasumi-1 and CD34+ cells (CEBPA; E) (F) Western blot analysis showing Gfi1b expressed from MSCV in Kasumi-1 cells. (G) FACS analysis of Kasumi-1 cells infected with MSCV-GFP or MSCV-Gfi1b-GFP at 3, 6, and 9 days following dTAG treatment (H and I) CUT&RUN analysis of LSD1 from Kasumi-1 cells. (H) Venn diagram showing overlap of LSD1 and AML1-ETO peaks. (I) LSD1 signal around HA-AML1-ETO peaks depicted in Fig. 3A before and after degradation of AML1-ETO-FKBP. (J) IGV screenshot of GFI1B showing the location of AML1-ETO and LSD1 peaks relative to active RNA polymerase measured by PROseq before and after treatment of with LSD1 inhibitor, dTAG-47 or the combination thereof. (K) Q-RT-PCR was performed to determine the relative expression of GFI1B compared to Actb control. Expression is shown relative to DMSO treated cells and error bars represent the RQmin and RQmax. (L) Flow cytometry detecting CD11B expression in parental Kasumi-1 cells or AML1-ETO-FKBP Kasumi-1 cells that were treated with dTAG-47 alone or in combination with 100 nM GSK2879552 and 1 μM ATRA.