MEN1 protein, human

ChIP was performed as previously described (28 (link)), using antibodies for Psip1/p75 (Bethyl laboratory A300–848), Psip1/p52 (Bethyl laboratory A300–847) Mll1 (Active Motif 61295), BMI1 (Millipore 05–637), Ring1B (MBL D139–3), H3K4me3 (Millipore 07–473), menin (Abcam, ab4452–50), RNA PolII Ser2p (Millipore 04–1571, Clone 3E10), Ctbp1 (Santa Cruz SC-55502), CBX4 (Abcam ab139815) and Mll2 (Abcam ab15962). ChIPed DNA was amplified with WGA2 using the manufacturer's protocol (Sigma Aldrich) and hybridized to custom Hox arrays (28 (link)). All ChIP on chip experiments were done with at least two biological replicates (GEO accession number GSE 49182 for platform GPL13276).
Normalization and analysis of microarray data was as described previously (28 (link)). For CpG analysis, CpG islands (CGIs) were identified by finding probes with a minimum of 25 bp overlap with CGI found ±1 kb within genes using Galaxy software. CGI positions were taken from the University of California Santa Cruz (UCSC) table browser.
Enrichment analysis for ChIP and run-on data was performed for probes ±1 kb from transcription start sites (TSS) or transcription end site (TES). The smoothed conditional mean plots were generated using the R package ggplot2 and the geom_smooth function.
The following mm9 coordinates were used for quantification of ChIP enrichment; non-expressed 3′ Hoax(Hoxa1 to Hoxa7) genes chr6:52,101,011–52,172,728, expressed 5′ Hoxa genes (Hoxa9-Hoxa13) chr6:52,171,296–52,211,033, 3′ non-expressed Hoxd (Hoxd1 to Hoxd9 genes) chr2:74,534,258–74,606,421, expressed 5′ Hoxd genes (Hoxd9-Hoxd13) chr2:74,484,916–74,537,448. To test the significance of differential ChIP enrichment at genomic regions a Wilcoxon rank-sum test was performed with a correction for multiple testing (Holm method) using the R statistical program.
For sequential ChIP (SeqChIP), antibodies were covalently coupled to Dynabeads with antibody coupling kit, (Invitrogen Cat. 14311D), using the manufacture's protocol. The first ChIP was eluted with 10 mM DTT and the elute was diluted 30 times with Radio-Immunoprecipitation Assay (RIPA) (50 mMTris, pH 7.5, 150 mMNaCl, 1% IGEPAL CA-630, 0.5% deoxycholate) buffer before continuing with the second ChIP. Primers used for ChIP qPCR are given in Supplementary Table S1.

The study was conducted in accordance with the principles expressed in the Declaration of Helsinki and approved by the Institutional Review Board of the University of Michigan (HUM00001441). Blood samples were obtained from healthy donors after written informed consent. Peripheral blood mononuclear cells (PBMC) were obtained by a standard Ficoll gradient (GE Healthcare). Monocytes were isolated from PBMC using anti-CD14 magnetic beads (Miltenyi Biotec) and cultured at a concentration of 0.5-1.0 x 10⁶ cells/ml in complete RPMI 1640 medium (Lonza) containing 10% FBS (Cell Generation), 100 U/ml penicillin, 100 µg/ml streptomycin (Mediatech), supplemented with either 50 ng/ml M-CSF or GM-CSF (R&D Systems). On day+3 fresh media containing the growth factors was added. On day+7 the cells were washed twice with PBS containing Ca²⁺ and Mg²⁺ (Lonza) and fresh complete RPMI (for resting MΦ), complete RPMI containing 100 ng/ml IFN-γ (for M1) or 10 ng/ml IL-4 and IL-13 (for M2) (all cytokines were from R&D systems) was added for 24h-72h. In some experiments 40 μM of the MLL-Menin inhibitor MI-2-2 at a 1:500 dilution (kindly provided by J. Grembecka and T. Cierpicki) [18 (link)] or vehicle DMSO (Sigma-Aldrich) was added at day+6 and M1 polarization was carried out on day+7 in the presence of the inhibitor or DMSO as described.