Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c

Abstract

Menin, the product of the Men1 gene mutated in familial multiple endocrine neoplasia type 1 (MEN1), regulates transcription in differentiated cells. Menin associates with and modulates the histone methyltransferase activity of a nuclear protein complex to activate gene expression. However, menin-dependent histone methyltransferase activity in endocrine cells has not been demonstrated, and the mechanism of endocrine tumor suppression by menin remains unclear. Here, we show that menin-dependent histone methylation maintains the in vivo expression of cyclin-dependent kinase (CDK) inhibitors to prevent pancreatic islet tumors. In vivo expression of CDK inhibitors, including p27 and p18, and other cell cycle regulators is disrupted in mouse islet tumors lacking menin. Chromatin immunoprecipitation studies reveal that menin directly associates with regions of the p27 and p18 promoters and increases methylation of lysine 4 (Lys-4) in histone H3 associated with these promoters. Moreover, H3 Lys-4 methylation associated with p27 and p18 is reduced in islet tumors from Men1 mutant mice. Thus, H3 Lys-4 methylation is a crucial function of menin in islet tumor suppression. These studies suggest an epigenetic mechanism of tumor suppression: by promoting histone modifications, menin maintains transcription at multiple loci encoding cell cycle regulators essential for endocrine growth control.

Fig. 1.

Pancreatic β cell hyperplasia, increased BrdUrd incorporation, loss of menin expression, and hypoglycemia in Men1^+/- mice. (A, B, D, and E) Immunostaining of BrdUrd (red) and insulin (green) in pancreatic tissue from 9-month-old Men1^+/+ (A and D) and Men1^+/- mice (B and E). (C) Quantification of BrdUrd⁺ cells in islets (n = 100) from Men1^+/+ and Men1^+/- mice. (F and G) Immunohistochemical detection of nuclear menin (red) and insulin (green) in pancreas from Men1^+/+ (F) and Men1^+/- mice (G). (H) Blood glucose level during random feeding in mice with the indicated age and genotype (wild-type or Men1^+/-). n = 8 or more animals for each genotype. After 20 weeks of age, hypoglycemia in Men1^+/- mice remained statistically significant at P < 0.01. (I) Glucose tolerance testing of 28-week-old wild-type (n = 11), and Men1^+/- littermates (n = 10) after 14-h overnight fast. Data in C, H, and I are presented as average ± SEM. [Original magnification: ×16 (A and B) and ×63 (D–G).]

Fig. 2.

Disrupted expression of genes encoding cell cycle regulators in Men1^+/- pancreatic islets. (A) Conventional RT-PCR analysis of mRNA isolated from Ficoll-gradient-purified pancreatic islets for the indicated markers. Levels of mRNA specifying p19 and actin (a loading control) are not detectably altered in Men1^+/- mice. (B) Quantitative RT-PCR on islets from 40-week-old Men^+/+ and Men1^+/- mice for the indicated markers. (C) Western blot comparing expression of the indicated proteins in purified islets from 40-week-old Men1^+/+ and Men1^+/- mice. Actin serves as a loading control. (D and E) Immunohistochemical detection of nuclear p27^Kip1 (black dots) reveals reduced expression in pancreatic islets of Men1^+/- mice. (F and G) Nuclear expression of the transcription factor Nkx6.1 (red) by insulin⁺ β cells (green) is maintained in Men1^+/- mice. (H and I) Nuclear expression of the transcription factor Islet-1 (red) by insulin⁺ β cells (green) is maintained in Men1^+/- mice. [Original magnification: ×40 (D and E) and ×63 (F–I).]

Fig. 3.

Menin promotes methylation of histone H3 associated with chromatin at the p18 and p27 genes. MIN6 cells were transfected with DNA specifying wild-type menin (wild-type), or mutant missense alleles of Men1 derived from human tumors that encode the amino acid substitutions A242V, L22R, or T344R. (A) Anti-menin ChIP was performed by using PP3 and PP4 specific for regions in the p18 promoter, or PP3 and PP4 specific for regions in the p27 promoter. Menin produced from alleles that retain histone methyltransferase activity (wild-type, L22R) associate with these promoters. (B) ChIP analysis using antibodies specific for trimethylated histone H3 Lys-4. (C and D) ChIP analysis using antibodies specific to menin and trimethylated histone H3 Lys-4 performed on isolated islets from Men1^+/+ and Men1^+/- mice. (E) Comparison of trimethylated histone H3 Lys-4 levels in Men1^+/+ and Men1^+/- islets by Western blotting. Actin protein served as a loading control.