ASH2L mediates USF1 interaction with the hSET1A core complex and Hox gene activation.

(A) Schematic representation of the HoxB4 promoter driven pREP4 luciferase episomal reporter construct. (B) K562 cells were transfected with a pREP4-hHoxB4-luc reporter, an expression vector for USF1, and siRNA targeting hSET1A, HCF1, ASH2L or PRMT1. A CMV-driven renilla luciferase plasmid was used as a transfection control. Transfected cells were cultured for 48 hrs and lysed for measurement of luciferase activity. Data are shown as mean ± SD. ** P<0.01; * P<0.05. (C) Western blotting analysis of the levels of ASH2L, hEST1A, PRMT1, and α-tubulin in K562 cells transfected with luciferase reporter and siRNA constructs. (D) ChIP analysis of USF1 binding, HCF1 recruitment, and H3K4me3 association in K562 cells transfected with the pREP4-hHoxB4-luc reporter, an expression vector for USF1, siControl, or siRNA targeting hSET1A. Data are shown as mean ± SD. ** P<0.01. (E)ASH2L of the hSET1A core complex directly interacts with GST-USF1. GST-USF1, pre-absorbed to glutathione-sepharose beads, was incubated with each Flag tagged component of the hSET1A complex and analyzed by WB with Flag antibody (Top). Expression of the hSET1A components purified from virally infected SF9 cells (Bottom). (F)Co-IP assay in K562 cells showing USF1 associates with hSET1A but not with MLL1. (G) Co-IP assay in K562 cells showing that the USF1 associated hSET1A complex does not contain CFP1. (H) Interaction of USF1 with hSET1A in the presence but not absence of ASH2L. GST-USF1 was pre-absorbed to glutathione-sepharose beads and incubated with the purified, baculovirus reconstituted hSET1A complex with or without ASH2L (Top). Immunoblots of the purified, baculovirus expressing hSET1A complex components with or without ASH2L are shown as inputs (Bottom).