Menin epigenetically represses Hedgehog signaling in MEN1 tumor syndrome

Abstract

Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumor syndrome that includes susceptibility to pancreatic islet tumors. This syndrome results from mutations in the MEN1 gene, encoding menin. Although menin acts as an oncogenic cofactor for mixed lineage leukemia (MLL) fusion protein-mediated histone H3 lysine 4 methylation, the precise basis for how menin suppresses gene expression and proliferation of pancreatic beta cells remains poorly understood. Here, we show that menin ablation enhances Hedgehog signaling, a proproliferative and oncogenic pathway, in murine pancreatic islets. Menin directly interacts with protein arginine methyltransferase 5 (PRMT5), a negative regulator of gene transcription. Menin recruits PRMT5 to the promoter of the Gas1 gene, a crucial factor for binding of Sonic Hedgehog (Shh) ligand to its receptor PTCH1 and subsequent activation of the Hedgehog signaling pathway, increases repressive histone arginine symmetric dimethylation (H4R3m2s), and suppresses Gas1 expression. Notably, MEN1 disease-related menin mutants have reduced binding to PRMT5, and fail to impart the repressive H4R3m2s mark at the Gas1 promoter, resulting in its elevated expression. Pharmacologic inhibition of Hedgehog signaling significantly reduces proliferation of insulinoma cells, and expression of Hedgehog signaling targets including Ptch1, in MEN1 tumors of mice. These findings uncover a novel link between menin and Hedgehog signaling whereby menin/PRMT5 epigenetically suppresses Hedgehog signaling, revealing it as a target for treating MEN1 tumors.

Figure 1

Menin regulates GAS1 and Hedgehog signaling. (A) Quantitative RT-PCR (qRT-PCR) and immunoblotting showing expression of Gas1 mRNA and protein in menin-null MEFs complemented with either vector or wild type (WT) menin. Ponceau S is included as a loading control. (B) qRT-PCR showing expression of Gli1 mRNA in menin-null MEFs complemented with either vector or WT menin cultured in varying concentrations of Sonic Hedgehog-conditioned medium (Shh-CM). Error bars indicate ± s.d.

Figure 2

Menin interacts directly with PRMT5 to methylate histone H4. (A) Immunoblotting of menin in nuclear extract (NE) from HEK293 cells expressing Flag-menin fractionated by anion-exchange chromatography using a Q-Sepharose column eluted with varying concentrations of NaCl. (B) Silver staining of menin-containing fractions after affinity purification using anti-Flag M2 beads. Visible bands were excised for identification by mass spectrometry. * = PRMT5 peptide fragments identified by mass spectroscopy; ⁷⁷DWNTLIVGK, ²²⁸AAILPTSIFLTNKK, ²⁴¹KGFPVLSK, ³³⁴YSQYQQAIYK, ³⁶⁹GPLVNASLR. (C) Immunoblotting showing menin (top), PRMT5 and MEP50 (bottom) in NE of HEK293 cells ectopically expressing Flag-menin immunoprecipitated with indicated antibodies. (D, E) Endogenous interaction between menin and PRMT5 in HEK293 cells immunoprecipitated with two independent anti-menin (D) or anti-PRMT5 antibodies (E). (F) Immunoblotting for menin, PRMT5 and MEP50 in gel filtration chromatography fractions of NE from HEK293 cells ectopically expressing Flag-menin. (G) Immunoblotting of His-menin expressed in E. coli immunoprecipitated by GST-PRMT5 immobilized to glutathione beads. (H) Biotinylated PRMT5 peptides of various lengths were immobilized on streptavidin beads, and the binding of His-menin was identified by immunoblotting. (I) Histone methyltransferase (HMT) assay using anti-menin immunoprecipitates from 293T cells transfected with Flag-menin and/or Myc-PRMT5 incubated with ³H-AdoMet and histone H4. Autoradiography for ³H-methylated histone H4 (middle); Coomassie stain of H4 is included as a loading control. (J) Immunoblotting of menin and PRMT5 in cells used for HMT assay in I. Ponceau S is included as a loading control.

Figure 3

Loss of PRMT5 results in elevated Gas1 levels and enhanced Hedgehog (Hh) signaling. (A) Quantitative RT-PCR (qRT-PCR) showing reduction of Prmt5 mRNA in MEFs expressing two independent Prmt5-targeting shRNA clones compared to MEFs expressing scrambled control shRNA. (B) qRT-PCR showing expression of Gas1 mRNA levels in MEFs expressing shRNA targeting Prmt5. (C) Immunoblotting for PRMT5 and GAS1 in MEFs expressing either control or Prmt5-targeting shRNA’s. Ponceau S is included as a loading control. (D) Chromatin immunoprecipitation (ChIP) with antibodies against PRMT5 and histone H4 arginine 3 symmetric dimethylation (H4R3m2s) at the Gas1 promoter. ChIP amplicon, Gas1 (−780 bp / −609 bp). (E–F) MEFs expressing control or two independent shRNA clones targeting Prmt5 were cultured in either control or Sonic hedgehog conditioned medium (Shh-CM) and Gli1 (E) and Ptch1 (F) mRNA levels were quantitated by qRT-PCR. Error bars indicate ± s.d.

Figure 4

Menin recruits PRMT5, and its associated histone modification mark, H4R3m2s, to the Gas1 promoter. (A) Chromatin immunoprecipitation (ChIP) with antibodies against menin, PRMT5, and histone H4 arginine 3 symmetric dimethylation (H4R3m2s) at the Gas1 promoter in Men1-null MEFs complemented with either empty vector or wild-type (WT) menin. ChIP amplicon, Gas1 (−780 bp / −609 bp). (B) Quantitative RT-PCR (qRT-PCR) showing expression of Gas1 mRNA in menin-null MEFs complemented with empty vector, WT menin or MEN1 disease related mutants, L22R and A242V. Levels of ectopic WT and mutant menin expression are shown, and immunoblotting for β actin is included as a loading control. (C) ChIP with antibodies against menin and H4R3m2s at the Gas1 promoter in Men1-null MEFs complemented with vector, WT menin or MEN1 disease related mutants, L22R and A242V. ChIP amplicon, Gas1 (−780 bp / −609 bp). (D) Co-immunoprecipitation of PRMT5 in menin immunoprecipitates from 293T cells ectopically expressing PRMT5, and either WT or MEN1 disease related menin mutants (top panel). Anti-menin immunoprecipitates from cells above were incubated with ³H-AdoMet and histone H4 for Histone Methyltransferase (HMT) assay, and ³H-methylated histone H4 was detected by autoradiography (third panel). Coomassie stain of H4 is included as a loading control (bottom panel). Error bars indicate ± s.d.

Figure 5

Loss of Men1 correlates with activated Hedgehog signaling in Men1-null mice. (A–D) Pancreatic islets were isolated from 8 mo. old Men1^l/l; RipCre and control Men1^l/l mice (n = 4 mice) and qRT-PCR was used to quantitate the mRNA levels of Men1, p < 0.0001 (A), Gas1, p < 0.0044 (B), Gli1, p = 0.0538 (C) and Ptch1, p = 0.0258 (D). Error bars indicate ± s.d.

Figure 6

Inhibition of Hedgehog (Hh) signaling in Men1-excised mice results in decreased islet cell proliferation. (A) Scheme for inhibition of the Hedgehog (Hh) pathway with GDC-0449 in Men1-excised mice. (B–C) Immunofluorescence for BrdU and insulin in pancreas of Men1-excised mice gavaged with either vehicle (B), or GDC-0449 (C) for four weeks at a dose of 100 mg/kg b.i.d. (D) Quantitation of BrdU incorporation in islets of mice above (same as B, C). (E) Quantitative RT-PCR (qRT-PCR) for Ptch1 mRNA in islets of Men1^l/l;pdxCreER mice gavaged for 10 days with either vehicle or GDC-0449 at a dose of 100 mg/kg b.i.d., p = 0.0010. (F) A model for menin-PRMT5-mediated inhibition of Hh signaling through epigenetic regulation of GAS1. Error bars indicate ± s.d.