Supplementary Materials1: Table S1. cancer. We assessed MLL/SET proteins and found that SETD1A is required for survival of acute myeloid leukemia (AML) cells. Mutagenesis studies and CRISPR-Cas9 domain screening, showed the enzymatic SET domain is not necessary for AML cell survival but that a newly identified region, termed the FLOS (Functional Location on SETD1A) domain, is indispensable. FLOS disruption suppresses DNA damage response genes and induces p53-dependent apoptosis. The FLOS domain acts as a Cyclin K-binding site that is required for chromosomal recruitment of Cyclin K, and for DNA repair-associated gene expression in S phase. These data identify a connection between the chromatin regulator SETD1A and the DNA damage response that is independent of histone methylation, and suggests that targeting SETD1A and Cyclin K complexes may represent a therapeutic opportunity for AML and potentially other cancers. In-brief Independent of its enzymatic activity, H3K4 methyltransferase SETD1A promotes leukemic cell survival by regulating DNA damage response. Open in a separate window Introduction Histone 3 lysine 4 methylation (H3K4me) is a post-translational modification that is associated with transcriptional regulation in multiple organisms from yeast to humans (Barski et al., 2007; Briggs et al., 2001; Ng et al., 2003; Shilatifard, 2008). The enzymes that catalyze this modification are important for the maintenance of developmental gene expression in multicellular organisms and are frequently mutated in human cancer (Bledau et al., 2014; Denissov et al., 2014; Ernst et al., 2004; Kandoth et al., 2013). H3K4 histone methyltransferases (HMTs) are defined by an enzymatic SET domain (Gu et al., 1992; Jones and Gelbart, 1993; Tschiersch et al., 1994). Yeast have a single H3K4 HMT, SET1, that is present in a multiprotein (COMPASS) complex responsible for all H3K4 methylation (Schneider et al., 2005). Mammals have 6 homologs of yeast SET1 that possess H3K4 HMT activity, including MLL1-4 (KMT2A-D) and SETD1A/B (KMT2F/G). Each of these enzymes is found in a multiprotein complex with overlapping subunits. While each of these complexes have been shown to possess H3K4 HMT activity, the extent to which their cellular functions are largely a result of SET-domain associated enzymatic activity is unclear. One of the most well characterized mammalian SET1 homologs is MLL1. MLL1 is found in fusion proteins generated by chromosomal translocation of the gene to one of more than 60 different partner genes (Krivtsov and Armstrong, 2007). Pediatric acute leukemia cases with and shRNAs suggested that the SETD1A/COMPASS complex might have critical roles in MLL-AF9 leukemia cells (Figure 1A). Efficacy and specificity of shRNAs were confirmed by rescue experiments with an exogenous SETD1A construct (Figure S1ACC). To evaluate the effect of knockdown in greater detail, we generated murine MLL-AF9 leukemia single cell clones which highly express a doxycycline inducible shRNA. After doxycycline administration, two shRNAs showed significant suppression of expression (Figure 1B), and inhibition of leukemia cell proliferation (Figure 1C). shRNAs order Nalfurafine hydrochloride induced apoptosis, cell cycle arrest, and neutrophilic differentiation (Figures 1D and 1E, S1D). To examine and compare the functional role of and on H3K4 methylation, we also performed an inducible knockdown of either or knockdown predominantly induced growth arrest and cell order Nalfurafine hydrochloride differentiation (Figures S1HCI). Surprisingly, knockdown did not show reduction of global H3K4me3 (Figure 1F). H3K4me3 ChIP-seq also showed no obvious change in H3K4me3 after knockdown of (Figure S1J). To confirm these effects on other leukemia cells, we suppressed in the MOLM-13 human leukemia cell line order Nalfurafine hydrochloride (Figures S1K). Consistent with murine cells, shRNA expression in MOLM-13 cells induced growth arrest and differentiation (Figures S1LCM). To examine a role for SETD1A in leukemia progression knockdown experiment in a MLL-AF9 murine leukemia model. At 3 weeks post-transplantation, shRNAs significantly suppressed the frequency of leukemia cells in the PB Rabbit Polyclonal to IRF4 (Figure 1G). In addition, shRNA constructs significantly prolonged the survival of recipient mice (Figures 1H). Consistent with data from the PB, knockdown strongly suppressed the growth of leukemia cells in bone marrow (Figure S1N). In mice that did get sick in the knockdown group, we did not observe GFP++ cells in their bone marrow (BM) (Figure S1O) demonstrating loss of shRNA expression. When.