AML1/ETO and AML1 interact with each other through the RHD domain of both proteins. (A) Structures of GST-AML1 fusion proteins used in the binding assays. The GST fusion proteins containing various forms of AML1 are schematically shown. The ability of the GST-fusion proteins to bind AML1/ETO is shown at right as + or −. (B) Autoradiography showing the binding of the ³⁵S-labeled AML1/ETO to various forms of the GST-AML1 fusion proteins. (C) Structures of the GST-AML1/ETO fusion proteins used in the binding assay. (D) Autoradiography showing the binding of the ³⁵S-labeled AML1 to the different truncated forms of the GST-AML1/ETO fusion proteins. The expression levels of the constructs used in B and D were almost equivalent, as shown in supplemental Figure 7. (E) Fluorescence micrographs in HEK-293T cells transfected with the indicated plasmids showing the in situ localization of AML1/ETO and AML1. AML1/ETO-∆RHD, AML1/ETO construct with the RHD domain deleted; AML1-∆RHD, AML1 construct with the RHD domain deleted; AML1-∆RHD-N, AML1 construct with most of the RHD domain deleted but the nuclear localization signal (NLS) within the RHD domain intact. Scale bars, 10 μm. (F) Validation for the RHD domain responsible for the association of AML1/ETO and AML1 on chromatin. ChIP was performed with anti-AML1 (C19) and anti-ETO antibodies in U937 cells transfected with either the full length AML1/ETO or AML1/ETO with the RHD domain deleted. There might exist 2 groups of the overlap regions: (i) AML1/ETO bound to chromatin regions prebound by wild-type AML1 and (ii) wild-type AML1 was recruited by AML1/ETO after AML1/ETO expression. The left panel shows the western blot validation for the expression of AML1/ETO and AML1/ETO-∆RHD transfected in U937 cells. NC, negative control. Error bars represent the SD of triplicate measurements.