AbstractThe H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain‐mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain‐binding partners reveals that the SETD1A FLOS domain binds mitosis‐associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3‐binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A‐bound promoter‐TSS regions and SETD1A‐negative H3K4me1‐positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A‐binding and leukemia cell proliferation. Cell‐cycle‐specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.