Fig 2: Identification of menin-associated proteins (MAPs) in breast cancer cells. (A) and (B) WES of BirA-Menin fusion proteins (A) and biotin-labeled proteins (B) in total lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after incubating with or without doxycycline and biotin. (C) Schematic purification and proteomic identification of MAPs using LC–MS/MS. (D) Heatmap of the quantification of 35 MAPs commonly shared in T47D and MCF-7 cells. MAPs further verified by WES immunoassays were indicated by arrows. (E) Network analysis of 35 MAPs in MCF-7 cells. The distance between menin and MAPs represented the quantitative ratio of each MAP and menin. MAPs marked in blue were further assayed by WES. (F) Nuclear or cytoplasmic lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after streptavidin beads pull-down were detected by WES with antibodies against menin, KMT2A, MED12, WAPL, GATA3, LaminA/C, or GAPDH. FL, full length; SP, spliced form.

Fig 3: USO1 depletion in transformed bone marrow cells shows reduced proliferation and colony forming potential. (A) Schematic of an in vitro model system to transform Lin- bone marrow cells from Cas9-egfp mice using overexpression of MLL-Af4 transgene in. (B) Analysis of overexpression of MLL-Af4 by RT-qPCR and western blot in retrovirally transduced Lin-Cas9MLL-Af4 cells, Lin-Cas9 cells were used as negative control and 70Z/3 cells transduced with MLL-Af4 were used as positive control. RT qPCR was performed with an optimized set of primers, normalized to L32, and represented as fold-change from an internal control for each experiment. Western Blotting was performed with an antibody to MLL1. Vinculin was used as a high molecular weight loading control. (C) Upper panel, schematic of murine Uso1 depletion experiments, showing location of sgRNAs relative to the gene, and RT-qPCR primer location. Bottom panel, western blot analysis of 70Z/3 cells transduced with three different sgRNAs targeting Uso1 (msg1-3) cloned in the MSCV.sgRNA.mCherry.v1 vector. (D) RT-qPCR analysis of Uso1 depletion in 70Z/3 cells. (E) MTS assay (Absorbance at 490 nm) to measure proliferation in Uso1-depleted 70Z/3 cells compared to NT control cells. (F) FACS plot showing gating schema to sort the Lin-Cas9MLL-Af4 GFP+ mCherry+ population following transduction with the Uso1 msg3 vector. (G) Western blot was used to confirm the reduction in USO1 expression in sorted Lin-Cas9MLL-Af4 cells. (H). Colony formation assay using Lin-Cas9MLL-Af4 cells in methylcellulose assay as described in methods with titration of input cell number (t test, ***P < 0.001; ****P < 0.0001).

Fig 4: RUNX3 defends against endogenous oncogenic K-Ras. a H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were synchronized by serum deprivation and stimulated with 10% serum and 1 μM 4-OHT for the indicated durations (0, 4, and 8 h). Time-dependent subcellular localization of the expressed proteins was analyzed by double immunofluorescence staining (green = RUNX3; red = F-actin). b, c H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were serum-starved for 24 h, stimulated with 10% serum or 10% serum + 1 μM 4-OHT. Cells were harvested at the indicated time points, and the time-dependent interactions of RUNX3 with BRD2, p300, H4K12-ac, TFIID complex (TAF1, TAF7, and TBP), SWI/SNF complex (BRG-1 and BAF155), and MLL1/5 were measured by IP and IB. Expression of p14ARF, p53, and p21 was measured by IB. The binding of the proteins and H4K12-ac, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. d H460-vec, H460-ERT2-RUNX3, and H460-ERT2-RUNX3-K94/171R cells were treated with indicated si-RNA, serum-starved for 24 h, and then stimulated with 10% serum or 10% serum + 1 μM 4-OHT for the indicated durations. Apoptotic cells were detected by flow cytometry after Annexin V–FITC/PI staining. The levels of p53 were measured by IB

Fig 5: RUNX1 Interacts with the AF4-MLL Complex and Activates Gene Targets(A) RUNX1 ChIP-seq in SEM cells compared with MLL-C:H3K4Me3 and MLL-N:AF4-C:H3K79Me2 ChIP-seq.(B–D) Sample ChIP-seq tracks from SEM cells across MEF2C (B), ADAM10 (C), and SPI1/PU.1 (D).(E) Gene expression analysis by real-time PCR in SEM cells treated with two different RUNX1 siRNAs (gray bars, siRNA#1; white bars, siRNA#2). For each experiment, the PCR signal was quantified relative to control-treated cells. Results represent the mean ± SD of three independent knockdown experiments.(F) Western blots as indicated in SEM cells treated with a nontargeting control, RUNX1 siRNA#1, or a wild-type MLL siRNA.(G) RUNX1 protein complex interactions. RUNX1 can interact with a wild-type AF4 complex (interaction 1), a wild-type MLL complex (interaction 2), and potentially with an AF4-MLL complex (interaction 3).(H and I) Immunoprecipitation (IP) experiments using RS4;11 (H) and SEM (I) nuclear extracts. Extracts were IP’d with αIgG (lane 2), αAF4-N (lane 3), αRUNX1 (lane 4) or αMLL-C (lane 5), blotted and probed with the antibodies indicated. Input lanes represent 1% of the amount of extract used for the IPs.(J) A schematic of the MEF2D, JUNB, JUND, and SPI-1 (aka PU.1) loci showing the approximate location of PCR primer sets (open arrow heads) used for ChIP analysis. Black box indicates consensus RUNX1 binding motifs in the upstream regulatory region (URE) of SPI-1 (Huang et al., 2008; Huang et al., 2011) and the first intron of MEF2D (Pencovich et al., 2011). Gray box indicates exon 1 of MEF2D, JUND, JUNB, and SPI-1.(K–M) ChIP analysis in SEM cells treated with a nontargeting control or RUNX1 siRNA#1 at the targets as indicated using antibodies to AF4-N (K), MLL-C (L), and RBBP5 (M). Error bars represent the ±SD of three separate PCR reactions.See also Table S5 and Figure S5.