MLL and menin copurify in a complex that regulates expression of CDK inhibitors. (A) Strategy for immunoprecipitating MLL complex from HeLa cells expressing FLAG-tagged WDR-5. N.E., nuclear extract. (B) Western blots showing that menin and mammalian homologues of yeast SET1 complexes copurify with MLL. IN, input; FT, flow-through; E, eluate. (C) Mll^-/- cells have a faster growth rate than Mll^+/+ cells or three independent Mll^-/- cells expressing FLAG-tagged MLL (F-MLL#1, F-MLL#6, and F-MLL#16). Growth curves were generated by plating cells at 1 × 10⁵ in T75 flasks and counting cells at the same time point in successive 24-h periods by using Trypan blue exclusion. (Inset) Higher levels of p27^Kip1 protein in Mll^+/+ (lane 1), F-MLL#1 (lane 3), F-MLL#6 (lane 4), and F-MLL#16 (lane 5) compared with Mll^-/- (lane 2) cell lines. (D) Menin-null cells (Men1^-/-) and null cells with an empty expression vector (Men1^-/- vec) have a faster growth rate than menin wild-type (Men1^+/+) cells and null cells expressing exogenous menin (Men1^-/- menin). (Inset) Higher p27^Kip1 levels in slower Mll^+/+ (lane 1), Men1^+/+ (lane 3), and Men1^-/- menin (5) compared with faster growing cells that are Mll^-/- (lane 2), Men1^-/- (lane 4), or Men1^-/- cells with an empty expression vector (lane 6). Western blot for menin shows lack of expression in Men1^-/- (lane 4) or Men1^-/- cells with an empty expression vector (lane 6) and restoration of menin expression in Men1^-/- menin cells (lane 5). (E) Real-Time PCR quantification using TaqMan probes shows higher expression of p27^Kip1 and p18^Ink4c transcripts in Mll^+/+ (dark blue), F-MLL#6 (gray), and F-MLL#16 (red) cell lines compared with Mll^-/- (green) cells. Gapdh and β-actin were both used as internal reference standards. TaqMan probe and primer sequences are available on request. (F) Quantitative RT-PCR shows higher expression of p27^Kip1 and p18^Ink4c transcripts in menin wild-type (Men1^+/+, dark blue) or null cells with menin reexpression (Men1^-/- men, red) compared with menin-null cells (Men1^-/-, green) or menin-null cells harboring an empty expression vector (Men1^-/- vec, gray). Cell lines that express patient-derived menin point mutants (L22R, black; A242V, light blue) show defective restoration of p27^Kip1 and p18^Ink4c expression (Fig. 2F Inset shows Western blot for menin expression in these cell lines).

Point mutations in menin interfere with binding and transcriptional regulation of CDK inhibitor loci. (A and B) Schematic of the p27^Kip1 (A) and p18^Ink4c (B) loci. Large arrow shows a major transcription start site at p27^Kip1 conserved in both mice and humans. Smaller arrows show minor transcription start sites. Black bars are CpG rich regions; gray bars are exons. Red bar represents TaqMan primer/probe set. The initial ATG is indicated. (C and D) ChIP with an anti-menin antibody quantified with Real-Time PCR. TaqMan primer and probe sequences are available on request. (C) Menin binds to the p27 coding region in cells that are Mll^+/+ (dark blue), Mll^-/- (green), Men1^+/+ (gray), or Men1^-/- with reexpressed menin (light blue), but not in Men1-null cells or at the Gapdh locus in any cell type (Men^-/-, red; Men1^-/- with an empty vector, black). (D) Menin binds to the p18^Ink4c coding region in cells that are Mll^+/+ (dark blue), Mll^-/- (green), Men1^+/+ (gray), or Men1^-/- with reexpressed menin (light blue). No binding is seen in Men1-null cells or at the Gapdh locus in any cell type (Men1^-/-, red; Men1^-/- with empty vector, black). (E) Luciferase assays on Men1^-/- cells cotransfected with menin expression vector and p27^Kip1 or p18^Ink4c luciferase reporter shows menin activates transcription from the p27^Kip1 or p18^Ink4c promoters (green versus blue bars). The L22R (gray) and A242V (red) mutants show impaired ability to activate transcription. (F) Binding of the menin mutants L22R (gray) and A242V (red) are reduced at the p27^Kip1 and p18^Ink4c loci compared with wild-type menin (Men1^-/- with reexpressed menin, green). (Inset) Western blot shows the L22R (gray lane 3) and A242V (red lane 4) mutants are expressed at similar levels to wild-type menin protein (green lane 2). Binding and menin expression in Men1^-/- cells with an empty vector are shown as controls.

MLL binding to CDK inhibitor loci is reduced in the absence of menin. (A) Western blots for Mll and menin expression in cell lines used for ChIP. Although Men^-/- cells (lane 4, red) express lower levels of Mll than Men1 wild-type cells (lane 3, gray), Men^-/- cells with empty expression vector (lane 6, black) or menin expression vector (lane 5, light blue) show comparable levels of Mll expression. men, menin; vin, vinculin loading control. For comparison, Mll expression is also shown in Mll^+/+ (lane 1, dark blue) and Mll^-/- cells (lane 2, green). (B) ChIP with anti-MLL^C antibody quantitated with Real-Time PCR shows that Mll binding to the p27^Kip1 coding region is much higher in cells expressing menin. (C) Mll binding to the p18^Ink4c coding region is also much higher in cells expressing menin. Binding to both p27^Kip1 and p18^Ink4c is high in Mll^+/+ (dark blue), Men1^+/+ (gray), or Men1^-/- with menin reexpression (light blue) compared with Men1-null cells (Men1^-/-, red), or Men1^-/- with an empty expression vector (black). Negligible binding is detected in Mll^-/- cells or at the Gapdh locus in any cell type.

MEN1 tumors show decreased p27^Kip1 expression compared with normal endocrine tissues. (A-F) Immunoperoxidase staining for menin, MLL, and p27^Kip1 in pancreatic tissues from a representative MEN1 patient. (A) Immunoperoxidase staining shows that menin is expressed in most morphologically normal islet cells. Expression is almost totally abolished in adenoma from the same tissue section (D). (B) MLL is expressed in essentially all normal islet cells. Expression is markedly decreased in adenoma from the same tissue section (E). (C) Immunoperoxidase staining for p27^Kip1 shows high-level expression in 97% of islet cells (500 nuclei count). Expression is decreased both in number of cells (82%; 500 nuclei count) and intensity of staining in adenoma from the same tissue section(F). (G) Plot of percentage of p27^Kip1-negative cells in normal islets and islet tumors based on 500 nuclei counts (n = 7). The blue horizontal bar represents average percent negative cells. (H) Model for role of MLL in normal homeostasis through regulation of Hox genes and CDK inhibitors (see text).