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Dynamic Epigenetic Regulation by Menin During Pancreatic ... ·...
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Chromatin, Gene, and RNA Regulation Dynamic Epigenetic Regulation by Menin During Pancreatic Islet Tumor Formation Wenchu Lin 1,2,3,4 , Hideo Watanabe 1,2,3 , Shouyong Peng 1,2,3 , Joshua M. Francis 1,2,3 , Nathan Kaplan 1,2,3 , Chandra Sekhar Pedamallu 1,2,3 , Aruna Ramachandran 1,2,3 , Agoston Agoston 2 , Adam J. Bass 1,2,3 , and Matthew Meyerson 1,2,3 Abstract The tumor suppressor gene MEN1 is frequently mutated in sporadic pancreatic neuroendocrine tumors (PanNET) and is responsible for the familial multiple endocrine neoplasia type 1 (MEN-1) cancer syndrome. Menin, the protein product of MEN1, associates with the histone methyltransferases (HMT) MLL1 (KMT2A) and MLL4 (KMT2B) to form menin–HMT complexes in both human and mouse model systems. To elucidate the role of methylation of histone H3 at lysine 4 (H3K4) mediated by menin–HMT complexes during PanNET formation, genome-wide histone H3 lysine 4 trimethylation (H3K4me3) signals were mapped in pancreatic islets using unbiased chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Integrative analysis of gene expression profiles and histone H3K4me3 levels identified a number of transcripts and target genes dependent on menin. In the absence of Men1, histone H3K27me3 levels are enriched, with a concomitant decrease in H3K4me3 within the promoters of these target genes. In particular, expression of the insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) gene is subject to dynamic epigenetic regulation by Men1-dependent histone modification in a time-dependent manner. Decreased expression of IGF2BP2 in Men1-deficient hyperplastic pancre- atic islets is partially reversed by ablation of RBP2 (KDM5A), a histone H3K4-specific demethylase of the jumonji, AT-rich interactive domain 1 (JARID1) family. Taken together, these data demonstrate that loss of Men1 in pancreatic islet cells alters the epigenetic landscape of its target genes. Implications: Epigenetic profiling and gene expression analysis in Men1-deficient pancreatic islet cells reveals vital insight into the molecular events that occur during the progression of pancreatic islet tumorigenesis. Mol Cancer Res; 13(4); 689–98. Ó2014 AACR. Introduction Multiple endocrine neoplasia type 1 (MEN-1) is an auto- somal-dominant syndrome, characterized by multiple tumors in endocrine tissues such as the pituitary gland, parathyroid gland, and pancreatic islets (1). Linkage studies and posi- tional cloning identified the causative gene, MEN1, for this disorder. Over 1,300 mutations, typically truncating, have been identified in MEN1 (2, 3). The importance of MEN1 inactivation in tumorigenesis is highlighted by the frequency of MEN1 mutations in sporadic endocrine tumors—44% in pancreatic neuroendocrine tumors and 35% in parathyroid adenomas (4, 5). Heterozygous and conditional Men1 knock- out mice develop tumors in multiple neuroendocrine tissues, recapitulating the spectrum of tumors in MEN-1 syndrome, with Men1 conditional knockout animals demonstrating a shorter latency (6–9). Although Men1 mutations are primarily associated with neuroendocrine cancers, several lines of evi- dence demonstrate that Men1 can also be dysregulated in non-neuroendocrine tumors such as lung cancer, melanoma, and liver cancer (10–12). Several studies have implicated that menin, the protein product of MEN1, is involved in transcriptional regulation, cell-cycle control, protein degradation, and genome instability through interaction with a number of transcription factors such as JunD, NF-kB, and members of the Smad family (3, 13–15). In addition, we and others have shown that menin is physically associated with Trithorax-like complexes containing the histone methyl- transferases MLL1 (KMT2A) and MLL4 (KMT2B, previously MLL2), to promote trimethylation of histone H3 at lysine 4 (H3K4me3; refs. 16, 17). Surprisingly, menin also binds to the MLL fusion protein in leukemia cells to upregulate HoxA9 gene expression, thus promoting oncogenic activity in MLL-associated leukemiogenesis (18). Genome-wide analysis by chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip; ref. 19) has revealed that menin colocalizes with MLL at gene promoters in various cell types, suggesting that menin regulates transcription in cooperation with MLL in multiple tissues (20). Currently, there are no targeted therapies directed toward patients harboring pancreatic neuroendocrine cancer tumors with MEN1 mutations. Thus, there is a critical need to deepen our understanding of the biology of these cancers to develop more effective therapeutic approaches. Given the inactivation of menin 1 Department of Medical Oncology, Center for Cancer Genome Dis- covery, Dana-Farber Cancer Institute, Boston, Massachusetts. 2 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts. 3 Cancer program, Broad Institute of Harvard and MIT, Cambridge, Massachusetts. 4 High Magnetic Field Laboratory, Chinese Academy of Sciences, 350 Shushanhu RD, Hefei, Anhui Province, 230031, P. R. China. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). W. Lin and H. Watanabe contributed equally to this article. Corresponding Author: Matthew Meyerson, Dana-Farber Cancer Institute, Boston, MA 02215. Phone: 617-632-4768; Fax: 617-582-7880; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-14-0457 Ó2014 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org 689 on November 25, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst December 23, 2014; DOI: 10.1158/1541-7786.MCR-14-0457
Transcript of Dynamic Epigenetic Regulation by Menin During Pancreatic ... ·...
Chromatin, Gene, and RNA Regulation
Dynamic Epigenetic Regulation by Menin DuringPancreatic Islet Tumor FormationWenchu Lin1,2,3,4, Hideo Watanabe1,2,3, Shouyong Peng1,2,3, Joshua M. Francis1,2,3,Nathan Kaplan1,2,3, Chandra Sekhar Pedamallu1,2,3, Aruna Ramachandran1,2,3,Agoston Agoston2, Adam J. Bass1,2,3, and Matthew Meyerson1,2,3
Abstract
The tumor suppressor gene MEN1 is frequently mutated insporadic pancreatic neuroendocrine tumors (PanNET) and isresponsible for the familial multiple endocrine neoplasia type 1(MEN-1) cancer syndrome. Menin, the protein product ofMEN1, associates with the histone methyltransferases (HMT)MLL1 (KMT2A) and MLL4 (KMT2B) to form menin–HMTcomplexes in both human and mouse model systems. Toelucidate the role of methylation of histone H3 at lysine 4(H3K4) mediated by menin–HMT complexes during PanNETformation, genome-wide histone H3 lysine 4 trimethylation(H3K4me3) signals were mapped in pancreatic islets usingunbiased chromatin immunoprecipitation coupled withnext-generation sequencing (ChIP-seq). Integrative analysis ofgene expression profiles and histone H3K4me3 levels identifieda number of transcripts and target genes dependent on menin.In the absence ofMen1, histone H3K27me3 levels are enriched,
with a concomitant decrease in H3K4me3 within the promotersof these target genes. In particular, expression of the insulin-likegrowth factor 2 mRNA binding protein 2 (IGF2BP2) gene issubject to dynamic epigenetic regulation by Men1-dependenthistone modification in a time-dependent manner. Decreasedexpression of IGF2BP2 in Men1-deficient hyperplastic pancre-atic islets is partially reversed by ablation of RBP2 (KDM5A), ahistone H3K4-specific demethylase of the jumonji, AT-richinteractive domain 1 (JARID1) family. Taken together, thesedata demonstrate that loss ofMen1 in pancreatic islet cells altersthe epigenetic landscape of its target genes.
Implications: Epigenetic profiling and gene expression analysis inMen1-deficient pancreatic islet cells reveals vital insight into themolecular events that occur during the progression of pancreaticislet tumorigenesis. Mol Cancer Res; 13(4); 689–98. �2014 AACR.
IntroductionMultiple endocrine neoplasia type 1 (MEN-1) is an auto-
somal-dominant syndrome, characterized by multiple tumorsin endocrine tissues such as the pituitary gland, parathyroidgland, and pancreatic islets (1). Linkage studies and posi-tional cloning identified the causative gene, MEN1, for thisdisorder. Over 1,300 mutations, typically truncating, havebeen identified in MEN1 (2, 3). The importance of MEN1inactivation in tumorigenesis is highlighted by the frequencyof MEN1 mutations in sporadic endocrine tumors—44% inpancreatic neuroendocrine tumors and 35% in parathyroidadenomas (4, 5). Heterozygous and conditional Men1 knock-out mice develop tumors in multiple neuroendocrine tissues,
recapitulating the spectrum of tumors in MEN-1 syndrome,with Men1 conditional knockout animals demonstrating ashorter latency (6–9). Although Men1 mutations are primarilyassociated with neuroendocrine cancers, several lines of evi-dence demonstrate that Men1 can also be dysregulated innon-neuroendocrine tumors such as lung cancer, melanoma,and liver cancer (10–12).
Several studies have implicated thatmenin, the protein productof MEN1, is involved in transcriptional regulation, cell-cyclecontrol, protein degradation, and genome instability throughinteraction with a number of transcription factors such as JunD,NF-kB, andmembers of the Smad family (3, 13–15). In addition,we and others have shown that menin is physically associatedwith Trithorax-like complexes containing the histone methyl-transferases MLL1 (KMT2A) and MLL4 (KMT2B, previouslyMLL2), to promote trimethylation of histone H3 at lysine 4(H3K4me3; refs. 16, 17). Surprisingly, menin also binds to theMLL fusion protein in leukemia cells to upregulate HoxA9 geneexpression, thus promoting oncogenic activity inMLL-associatedleukemiogenesis (18). Genome-wide analysis by chromatinimmunoprecipitation coupled with DNA microarray analysis(ChIP-chip; ref. 19) has revealed that menin colocalizes withMLL at genepromoters in various cell types, suggesting thatmeninregulates transcription in cooperation with MLL in multipletissues (20).
Currently, there are no targeted therapies directed towardpatients harboring pancreatic neuroendocrine cancer tumorswithMEN1 mutations. Thus, there is a critical need to deepen ourunderstanding of the biology of these cancers to develop moreeffective therapeutic approaches. Given the inactivation of menin
1Department of Medical Oncology, Center for Cancer Genome Dis-covery, Dana-Farber Cancer Institute, Boston, Massachusetts.2Department of Pathology, Brigham and Women's Hospital andHarvard Medical School, Boston, Massachusetts. 3Cancer program,Broad Institute of Harvard and MIT, Cambridge, Massachusetts.4High Magnetic Field Laboratory, Chinese Academy of Sciences,350 Shushanhu RD, Hefei, Anhui Province, 230031, P. R. China.
Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).
W. Lin and H. Watanabe contributed equally to this article.
Corresponding Author: Matthew Meyerson, Dana-Farber Cancer Institute,Boston, MA 02215. Phone: 617-632-4768; Fax: 617-582-7880; E-mail:[email protected]
doi: 10.1158/1541-7786.MCR-14-0457
�2014 American Association for Cancer Research.
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in multiple endocrine cancers and the reversibility of histoneH3K4me3, we were interested in the role of enzymes that poten-tially antagonize the histonemethylation activity of menin. Rbp2(Kdm5a, Jarid1a), initially identified as Retinoblastoma-bindingprotein 2, is a member of the Jumonji (JMJ) domain-containingfamily of histone demethylases, with roles in chromatin modi-fication and transcriptional regulation (21). Loss of Rbp2 recruit-ment to the CDKN1B gene is highly correlated with increasedhistone H3K4me3 levels and elevated gene expression (21–23).We have previously demonstrated in murine models that inacti-vation of the histone demethylase Rbp2 significantly inhibitstumor growth inMen1-deficent mice (24). We also demonstratedthat alterations in gene expression patterns upon Men1 loss inpancreatic islets are partially reversed by Rbp2 loss in these cells(24). Collectively, these observations support the notion that (i)histone methylation plays a key role in Men1 deletion-mediatedtumorigenesis in neuroendocrine cells, and (ii) the demethylaseenzyme activity of Rbp2 antagonizes the histone methyltransfer-ase activity associated with menin at gene loci such as CDKN1B.Although loss-of-function of menin is known to play an impor-tant role in tumor initiation and progression in endocrine tissues(7), there is limited information on the mechanisms linkingmenin–HMT complexes to neuroendocrine-specific hyperplasiaand tumorigenesis.
Men1-deficient mice can take up to a year to accumulate thenumerous genetic and epigenetic alterations that result in tumorformation, a slow process during which pancreatic islet cellstransform from normal to a hyperplastic and finally a malignantstate (7). Thus, this period represents a window of opportunity toinvestigate early events leading to tumorigenesis and to addressthe role of menin–HMT complexes in modulating cell prolifer-ation and behavior at this precancerous stage.
To investigate tumor formation mediated by alterations inH3K4me3 levels and to identify gene targets of menin–HMTcomplexes, we conducted epigenetic profiling of Men1-deficentpancreatic islets in 2-month-oldMen1 conditional knockoutmiceand control wild-type littermates, Using ChIP techniques coupledwith next-generation sequencing (ChIP-seq), we found thatMen1loss lowered H3K4me3 levels at select target gene promoters,resulting in downregulation of gene expression. In addition, lossof H3K4me3 correlated with increased H3K27me3 levels, con-sistent with the known association of H3K27me3 with generepression (25). Our study is the first to identify gene targets ofmenin–HMT complexes in mouse pancreatic islets in vivo alongwith the time course of the epigenetic changes accompanyingmouse pancreatic neuroendocrine tumor formation.
Materials and MethodsMouse experiments
Creation and genotyping of RIP-Cremice,Men1 KO mice, andMen1/Rbp2 KO mice has been described previously (24). Micewere maintained on a mixed 129s6, FVB/N, and C57BL/6 back-ground. All procedures were carried out in accordance with theNational Research Council Guide for the Care and Use of Lab-oratory Animals and were approved by the Institutional AnimalCare and Use Committee of the Dana-Farber Cancer Institute(Boston, MA).
Isolation of mouse pancreatic isletsPancreatic islets were isolated as previously described (24).
Histologic and immunohistochemical analysis for pancreatictissues
Pancreata were collected from mice at indicated time pointsand fixed in 4% paraformaldehyde for 2 hours followed bydehydration and paraffin embedding. Histopathologic analysiswas carried out on 5 mm sections stained with hematoxylin andeosin. Islet morphology and tumors were examined in at leastthree cut sections for each pancreas after staining with hematox-ylin and eosin. Appropriate positive and negative controls wererun on matched sections for all applied antibodies. Immunohis-tochemical staining was performed on serial sections using anti-bodies against H3K4me3 (Active Motif, catalog no. 36159,1:500), H3K27me3 (Cell Signaling Technology, catalog no.9733, 1:100) and Igf2bp2 (Abcam, Ab124930, 1:1,000). Sectionswere counterstained inMeyer hematoxylin, mounted, and photo-graphed using an Olympus microscope.
ChIP-seqFor each ChIP experiment, islets from at least 4 adult mice
were purified by collagenase digestion and gradient centrifuga-tion, with subsequent hand picking. ChIP was performed asdescribed (26) using 4 mg of anti-H3K4me3 (ActiveMotif, catalogno. 36159) or anti-H3K27me3 antibodies (Cell Signaling Tech-nology, catalog no. 9733). Five to 50 ng of DNA was used forlibrary construction. DNA was prepared for sequencing by Illu-mina cluster generation using a SPRI-work system with 100–300bp size selection followed by enrichment with barcoded PCRprimers for multiplexing. Sequencing was performed on aHiseq2000 machine for 40 nucleotides from a single end, at theMIT BioMicro Center. Barcode-separated FASTQ files were gen-erated from QSEQ files.
ChIP-Seq data analysisForty nucleotides of sequenced readswere aligned to themouse
reference genome (mm9 assembly), using Bowtie aligner (27).Only those reads/tags that mapped to unique genomic locationswith at most two mismatches were retained for further analysis.Histone mark peaks were detected using MACS (version 1.4.2) aspreviously described (28), with a P value cutoff of 10�6 andwith default values for other parameters. Quantitative changesin H3K4me3 upon Men1 deletion was assessed using MAnormalgorithm (29) with a P value cutoff of 10�5 and >1 log2 foldchange. H3K4me3 peaks that significantly decreased in Men1-deficient comparedwithRIP-Cre control isletswere assigned to themost adjacent gene within 30 kb; these genes were assessed foroverlap with genes downregulated in Men1-deficient mice.H3K4me3 ChIP signals were plotted around transcription startsites (TSS)of bothupregulated anddownregulatedgenes inMen1-deficient islets. H3K27me3 ChIP signals were plotted around thepeaks of either unchanged or decreased levels of H3K4me3 inMen1-deficient compared with RIP-Cre control islets.
Data availabilityAll ChIP-Seq data generated in this study have been deposited
in the NCBI GEO repository (accession number GSE63020).
RNA isolation and qRT-PCRTotal RNAwas isolated using theRNeasyKit (Qiagen) from100
to 300mouse pancreatic islets purified from 2mice with differentgenotypes. RNA quality was assessed on the Agilent Bioanalyzer.
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For RT-PCR, DNase I (Qiagen)-treated RNA samples were reversetranscribed using oligo-dT and SuperScript III (Invitrogen), withfirst strand cDNA used for PCR using SYBR Green PCR Mix(Qiagen) in an Applied Biosystems 7300 Real Time-PCR system.Standard ChIP with H3K4me3 and H3K27me3 antibodies wasperformed on mouse pancreatic islets in duplicate. PCR primerpairs were designed to amplify 150- to 200-bp fragments fromselect genomic regions. Primer sequences are listed in Supple-mentary Table S5.
ResultsInactivation of Men1 does not alter global H3K4me3 levels inpancreatic beta cells
To address whetherMen1 loss causes global changes in histoneH3K4methylation, we assessedH3K4me3 levels inMen1-deficientmouse pancreatic islets and control RIP-Cre islets by immunohis-tochemistry (IHC).Wedetectedno significant change in the overalllevels of H3K4me3 in Men1-deficient islets compared with wild-type islets (Fig. 1A). Our finding is consistent with earlier studiesdemonstrating that in contrast to Set1a and Set1b, themajorH3K4trimethylases inmammalian cells (30),MLL1andMLL4, theHMTsknown toassociatewithmenin, are responsible forH3K4 trimethy-lationof only a subset of loci (31).Ourobservations are also in linewith studies showing that MLL1 loss decreasedH3K4me3 levels inless than 5% of genes inmouse embryonic fibroblasts (MEFs) andthat MLL4 knockdown had no impact on overall H3K4me3 levelsin mouse embryonic stem cells (31, 32). We previously reportedthat inactivation of Rbp2 could partially rescue the tumor pheno-type inMen1-deficientmice (24). Immunohistochemistry revealedno appreciable change in overall H3K4me3 levels in Men1/Rbp2
double knockout islets comparedwithwild-type orMen1-deficientislets (Fig. 1A, right panel), suggesting thatmenin-HMT complexespotentially regulate histone modifications for only a subset ofgenes in mouse pancreatic islets.
Menin-dependent H3K4me3 is altered during early stages ofpancreatic neuroendocrine tumor formation
To investigate locus-specific H3K4 trimethylation potentiallyregulated by menin-HMTs in pancreatic islets, we performedChIP-seq to identify genome-wide H3K4me3 occupancy in isletsfrom2-month-oldRIP-Cre (control)mice orMen1-deficientmice.We identified >22,000 H3K4me3 peaks, using the MACS algo-rithm (28). As expected, H3K4me3 marks were most frequentlyobserved at proximal promoter regions, near TSSs; ref. 16; Sup-plementary Tables S1 and S2). Comparable profiles wereobserved in two independently purified batches of islets fromRIP-Cre mice (data not shown). We next compared H3K4me3signals inMen1-deficient pancreatic and control islets. Consistentwith our predictions from the IHC results described above, weobserved differential H3K4me3 signals only in a subset of regions(Fig. 1B). Among the total 1565 differential peaks identified incontrol cells (P < 10�5, fold change >2), only 815 peaks (�3.5%of total H3K4me3 peaks) showed a decrease in H3K4me3 signalsin Men1-deficient islets (Fig. 1B, and Supplementary Table S3),representing potential menin-HMT targets.
Integrative analysis of H3K4 trimethylation and geneexpression identifies menin-HMT gene targets
To determine whether the regulation of differentially expressedgenes in Men1-deficient islets is dependent on menin-mediatedH3K4me3, we integrated gene expression data (24) with
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Figure 1.H3K4me3 profiles in mousepancreatic islets. A, total H3K4me3levels were evaluated in pancreaticislet cells by IHC. Islets were purifiedfrom 2-month-old mice in which Men1was deleted using an islet-specific Credriver, either alone (Men1f/f; RIP-Cre)or in combination with Rbp2 (Men1f/f;Rbp2f/f; RIP-Cre). Islets from micebearing the Cre driver alone (RIP-Cre)were used as controls. B, volcano plotillustrating differential changes inH3K4me3 levels in Men1�/� isletsversus wild-type controls (left, greendots represent decreases in the Men1KO while red dots representincreases). Heatmap of those loci fromthe volcano plot showing significantalterationsof H3K4me3 levels inMen1-null islets compared with wild-typeislets (right).
Epigenetic Regulation by the Men1 Tumor Suppressor Gene
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H3K4me3 profile data. Genes whose expression was downregu-lated at least two-fold inMen1-deficient islets showed a significantreduction in H3K4me3 levels (Fig. 2A). In contrast, H3K4me3levelswere unchanged at genes upregulated at least two-fold uponmenin loss (Fig. 2B). Notably the H3K4me3 signal in down-regulated loci was also generally lower than for genes showingincreased expression uponMen1 loss (compare profiles of Fig. 2Aand B). These data suggest a direct role for menin-dependentH3K4me3 in activating gene expression from its target promotersin mouse pancreatic islets. The role of H3K4me3 in menin-dependent repression of gene expression however remains to beelucidated.
To identify genes that were downregulated and associated withreduced H3K4me3 levels in Men1-deficient islets we intersectedthese two datasets, revealing that 50 out of 365 genes showingreduced gene expression (14% overlap rate vs 1.7% expectedrandom overlap rate; P < 0.001) also exhibited lower H3K4me3levels (Fig. 2C). Many of these targets are linked to type 2 diabetesand beta cell functions (also see Supplementary Table S4), withIgf2bp2 (insulin-like growth factor 2 mRNA binding protein 2)emerging as one of the most significant genes in this category. Incontrast, of the 1227 genes that showed increased expression
upon Men1 loss, only 40 (3.1% overlap rate vs 5.7% expectedrandom overlap rate; not enriched) showed a correspondingincrease in H3K4me3 (Supplementary Fig. S1). These data indi-cate that gene repression mediated by menin under wild-typeconditions likely occurs by mechanisms other than via reductionof H3K4 tri-methylation.
Men1-loss does not change histone modifications at knownmenin gene targets in mouse pancreatic islets, at age of 2months
A number of genes including Cdkn1b, Cdkn2c, Mnx1, Hoxa9,andHoxc6, have been previously shown to be regulated bymeninor menin-HMT complexes (16, 18, 20, 33, 34). We, however, didnot observe a significant difference in enrichment of H3K4me3 orreduction of H3K27me3 at the Cdkn1b, Cdkn2c, and Mnx1promoters in Men1-deficient islets (Supplementary Fig. S2),despite Cdkn1b and Cdkn2c being highly expressed in mousepancreatic islets (WL, unpublished observations). These find-ings, although consistent with our prior observations (24)where we reported no significant difference in expression ofthese genes upon Men1 loss, are at odds with other studiesusing mouse embryonic fibroblasts (MEF) and pancreatic islets
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Figure 2.H3K4me3 levels are significantlydecreased in genes downregulated inMen1-deficient islets. H3K4me3 levelswere assessedwithin a 3 kbwindowofthe TSS of genes up- ordownregulated in Men1-depleted isletcells compared with wild-type. A, asignificant reduction in H3K4me3signal was observed in genes thatwere downregulated inMen1KO islets.B, no difference was apparent in thepromoters of genes upregulated inMen1 KO islets. C, overlap of genesshowing decreased expression inMen1 KO islets with those showingreduction in H3K4me3 levelsidentified 50 common targets.
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that have demonstrated the role of trimethylation of H3K4 intranscriptional activation of Cdkn1b and Cdkn2c (34). It ispossible that expression of Cdkn1b and Cdkn2c does not requiremenin–HMT complexes at this stage.
Menin is also known to regulate the majority of Hox genes inMEFs (31).Hox genes are typically bivalently modified in embry-onic stem cells (32); however, we did not observe strongH3K4me3 signals at these loci in pancreatic islets (SupplementaryFig. S3) although we detected strong H3K27me3 association withfour Hox gene clusters (Supplementary Fig. S3). As H3K27me3 isassociated with repression of gene expression, our results areconsistent with microarray data showing that most Hox geneswere silenced or expressed at very low levels (data not shown).Thus, at the time point being evaluated, menin-driven geneexpression appears to occur via both H3K4me3-dependent and-independent mechanisms.
Loci with increased H3K27me3 signals inMen1-deficient isletsare associated with decreased H3K4me3
Trimethylation of histone H3K4 is mediated by trithoraxgroup proteins including MLL1 and MLL4 and can antagonizeH3K27 trimethylation mediated by polycomb group com-plexes such as PRC2 (35, 36). To investigate this potentialinverse correlation, we evaluated H3K27me3 levels by IHC incontrol, Men1 single knockout and Men1/Rbp2 double knock-out islets. As observed for H3K4me3, we did not detect signif-icant differences in overall expression of H3K27me3 (Fig. 3A).We next asked whether loss of H3K4me3 in genes regulated bymenin–HMT complexes altered H3K27me3 levels. We com-pared H3K27me3 signals on H3K4me3-marked regions classi-fied as unchanged or decreased byMen1 loss (Figs. 1B, gray andgreen points and 3B). We detected enrichment of H3K27me3at promoters that showed decreased levels of H3K4me3 in
D H3K27me3 profile centered around H3K4me3 peaks in Men1 KO islets
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100 μm 100 μm 100 μmFigure 3.Increased H3K27me3 levels areobserved in regions with decreasedH3K4me3. A, H3K27me3 levels wereevaluated in pancreatic islet cells byimmunohistochemical analysis. Isletswere purified frommice in whichMen1was deleted using an islet-specific Credriver either alone (Men1f/f; RIP-Cre)or in combination with Rbp2 (Men1f/f;Rbp2f/f; RIP-Cre). Islets from micebearing the Cre driver alone (RIP-Cre)served as controls. B, regions ofH3K4me3 occupancy were classifiedaccording to changes in baselinelevels occurring uponMen1 loss. C andD, H3K27me3 levels were assessedwithin a 20 kb window spanning thecenter of the H3K4me3 signal in lociexhibiting decreased H3K4trimethylation (red trace) or nochange (purple) in RIP-Cre (C) orMen1-depleted (D) islet cells. Asignificant enrichment in H3K27me3signal was observed in genesassociated with decreased H3K4me3level in Men1 KO cells (red peaks inarea defined by green dotted lines inD), but not in wild-type cells (C).
Epigenetic Regulation by the Men1 Tumor Suppressor Gene
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Men1-deficient islets, but not at promoters with unalteredH3K4me3 occupancy (compare red tracings in Fig. 3C andD). In total, 37 of 50 genes with both decreased expressionand decreased H3K4me3 in Men1-deficient islets showedenhanced H3K27me3 (Supplementary Fig. S4).
Dynamic time-dependent changes in H3K4me3 andH3K27me3 at the Igf2bp2 promoter
Next, we sought to establish how H3K4me3 and H3K27me3signals change over time during pancreatic islet tumorigenesis.We focused on the Igf2bp2 locus as Igf2bp2 was one of the twomost downregulated genes in Men1-deficient islets comparedwith RIP-Cre islets based on analysis of microarray data, andbecause the Igf2bp2 promoter showed the most dramaticchange in H3K4me3 signal, of the 50 genes identified in ourintegrative analysis. We isolated pancreatic islets from controland Men1-deficient mice at 2, 6, and 12 months of age andtumors from Men1-deficient mice at 12 months. ChIP-seq toassess changes in H3K4me3 and H3K27me3 signals over time(Fig. 4A) showed, at 2 months, that H3K4me3 signals at theIgf2bp2 promoter in RIP-Cre (control) islets (shown in blue)localized to two peaks; upstream of exon 1 (Peak 1) andbetween exons 1 and 2 (Peak 2). In Men1-deficient islets, both
peaks were decreased, with Peak 2 showing a greater reduction.At 6 months, H3K4me3 signals were reduced in both controland Men1-deficient islets compared with 2 months. Finally, at12 months we observed a further reduction in H3K4me3 levelsin wild-type mice and in tumors from Men1-deficient animals,with a barely detectable Peak 2 in tumors (Fig. 4A).
In contrast, H3K27me3 occupancy at the Igf2bp2 promoterexhibited an inverse trend over time. We detected no appreciableH3K27me3 signal in RIP-Cre islets from 2-month-old mice,whereas Men1-deficient islets showed enhanced H3K27me3, inregions distinct from H3K4me3–occupied areas. We observeda slight increase in H3K27me3 signals in the RIP-Cre isletsand strong H3K27me3 signals in the Men1-deficient islets from6-month-old mice, including an overlap with H3K4me3 Peak 2.This trend continued at 12 months, where we observed furtherenhancement of the H3K27me3 signal in control islets anda robust H3K27me3 signal in Men1-deficient tumors from12-month-old mice, spreading over the Igf2bp2 promoter,upstream regulatory and coding regions (Fig. 4B). Many othergenes also show a similar inverse relationship betweenH3K4me3and H3K27me3 signals that was consistent during the progres-sion of pancreatic islet tumorigenesis; examples include Gata6and Oxtr (Supplementary Fig. S5).
Igf2bp2
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Figure 4.Time-dependent epigenetic changesat the Igf2bp2 promoter. A, H3K4me3levels progressively decreasewith age in Men1�/� islets (red tracks)relative to RIP-Cre islets (blue tracks).c.p.m, counts per million mappedreads. B, H3K27me3 levels are higherin Men1�/� islets (red) than inRIP-Cre islets (blue) in 2-month-oldmice and continue to increase at6 and 12 months.
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Igf2bp2 expression is epigenetically regulated during pancreaticislet tumor formation
Analysis of our previously published microarray data demon-strated that Igf2bp2 expression is downregulated inMen1-deficentpancreatic islets and is partially restored inMen1�/�;Rbp2�/� islets(Fig. 5A). We verified these observations by quantitative PCR(qPCR) analysis, confirming that Igf2bp2 expression wasdecreased by 60% upon Men1 ablation and rescued to near-baseline levels in the Men1�/�;Rbp2�/� condition (Fig. 5B).ChIP-PCR revealed that H3K4me3 levels at the Igfbp2 locus inMen1-deficient islets decreased to <60% of control (consistentwith the ChIP-seq data) and was restored to baseline inMen1�/�;Rbp2�/� islets, whereas H3K4me3 signals in control regions (p53and Gapdh) were unaltered (Fig. 5C). To correlate the changes inIgf2bp2 mRNA expression with protein levels, we evaluatedIgf2bp2 expression by immunohistochemistry (IHC) in pancreaticislets. We detected a marked reduction in Igf2bp2 protein inMen1-deficient islets compared with control RIP-Cre (Fig. 5D).Consistent with transcript levels, Igf2bp2 protein levels were
restored in Men1�/�;Rbp2�/� islets. Collectively, these datareveal that Igf2bp2 is epigenetically regulated by menin–HMTcomplexes in mouse pancreatic islets and that epigeneticchanges occurring as a consequence of Men1 loss are partiallyrestored by ablation of the Rbp2 histone demethylase.
DiscussionMenin–HMT targets associated with tumor progression
Few genes to date have been established as targets of menin;these include Hoxc6, Hoxc8, Hoxa9, Cdkn2c, Cdkn1b, and Mnx1(16, 20, 33, 34). Menin is known to associate with a number ofhistone methyltransferases including MLL1 and MLL4 (16, 17).Here, we have investigated menin-induced epigenetic modifica-tions during pancreatic neuroendocrine tumorigenesis. Wesought to identify direct targets of menin–HMT complexes inmouse pancreatic islets via integrative analysis of ChIP-seq andgene expression data and have identified a number of genesregulated by menin-mediated H3K4 trimethylation in pancreatic
Men1 ;RIP-cref/fRIP-cre Men1 ;Rbp2 ;RIP-cref/f
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Figure 5.Igf2bp2, a menin-HMT target, isepigenetically regulated in mousepancreatic islets. A, relativeexpression of Igf2bp2 in islets fromwild-type, Men1�/�, and Men1�/�;Rbp2�/� mice as determined bymicroarray analysis, and subsequentvalidation by qPCR (B). C, ChIP-qPCRshowing H3K4me3 levels at theIgf2bp2, Trp53, and Gapdh TSS incontrol, Men1�/� orMen1�/�;Rbp2�/�
islet cells. Data are themean� SEM ofthree independent experiments.�� , P < 0.01. D, detection of Igfbp2 byIHC in wild-type, Men1�/� andMen1�/�; Rbp2�/� pancreatic isletsin 2-month-old mice.
Epigenetic Regulation by the Men1 Tumor Suppressor Gene
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islets. A similar approachhasbeen employedbyothers (37), usingmouse embryonic stem cells and mouse pancreatic islet-likeendocrine cells (PILEC) as models to study menin-mediatedH3K4me3 during ES cell differentiation. Consistent with ourobservations, the authors did not observe a correlation betweendownregulation of gene expression and decreased H3K4me3 inES cells uponMen1 loss. However, upon differentiation of ES cellsinto PILEC, decreased H3K4me3 levels were associated withdownregulated genes, indicating a role for menin–HMT com-plexes during ES cell differentiation and lineage specificationupon Men1 loss (37).
In contrast toprevious studies indifferent experimental systems(33, 34), we did not find trimethylation of H3K4 at the Cdkn2cand Cdkn1b genes to be dependent on menin–HMTs. Thus,regulation of Cdk inhibitor genes at this stage may be driven byother mechanisms or may be independent of menin. Menin hasalso been reported toplay a critical role during embryogenesis andMLL1-mediated leukemogenesis via regulation of Hox geneexpression (31).
Although several previous studies have focused on chromatinand gene expression targets of menin (20, 37), we have identifieddifferent targets in our study. Previous studies utilized differentexperimental systems, either nonhyperplastic islets (20) asopposed to the hyperplastic islets in our study, orMen1-deficientmESCs and mouse pancreatic islet-like endocrine cells(PILEC; 37). Furthermore, in studies of mouse embryos, wepreviously identified HoxC6 and HoxC8 as menin targets (16),but did not find these genes as targets in the current study,suggesting the possibility that Hox genes might be the targets ofmenin during embryonic stages rather than in adult stages.Indeed, in adult mouse pancreatic islets, we did not observesignificant expression of Hox genes or significant trimethylationof H3K4me3 at Hox gene promoters but did observe high levelsof H3K27 methylation at Hox gene promoters (SupplementaryFig. S3). Thus, we believe that the differences between the current
and previous studies are likely to represent the effect of Men1under different biologic circumstances, although we cannotexclude the possibility that some of these differences representthe effects of variation in experimental conditions. Furtherindependent studies in consistent cell types and tissues will berequired to clarify this issue in the long term.
Anticorrelation between H3K4me3 and H3K27me3 signalsWe observed that H3K27me3 signals were enriched in regions
showing decreased H3K4me3. This may arise from either amixedpopulation of individual cells with each feature or represent trulybivalent domainswithin a single cell. In the former case, this likelyreflects an indirect effect of Men1 loss as menin has not beenknown to associate with any histone K27 demethylase. However,as decreased gene expression is accompanied by decreasedH3K4me3 at menin target gene promoters (Fig. 2A), it is con-ceivable that these epigenetic marks may coexist within a givencell.
Genes regulating cell fate decisions during embryonic devel-opment are often characterized by dual H3K4me3 andH3K27me3 marks (38), a bivalent mark indicating a "poised"state which can either be activated or repressed during lineagespecification. The MLL4 methyltransferase that interacts withmenin (16, 17) has been reported to regulate bivalent promo-ters in mouse embryonic stem cells (32). During pancreatic isletcell differentiation some bivalent marks may keep genes silentand poised, capable of switching to either an activated orrepressed state in response to the appropriate signal, genesassociated with H3K4me3 alone, however, are inactivated byadditional trimethylation of H3K27 (ref. 38; Fig. 6). To ourknowledge, this is the first study providing evidence that asignificant number of menin-dependent mouse genes are sub-ject to bivalent histone modification and regulation underphysiologic conditions.
OFF
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Figure 6.Proposedmodel forepigenetic regulationbymenin andeffectongeneexpression.Histonemarksandexpression status ofgenes regulatedbymenin–HMTcomplexes inwild-type (top) and Men1-null (bottom) mouse pancreatic islets. Increased H3K4me3 marks mediated by menin–HMT complexes opens chromatin, allowingfor binding of promoter-specific transcription factors (44), general transcription factors (GTF) and RNA polymerase II (Pol II), to facilitate gene expression. Loss of theH3K4me3 mark due to deletion of Men1, along with addition of the repressive H3K27me3 mark, possibly by PRC2 (Polycomb Repressive Complex 2) complexes,results in closed chromatin and repression of gene expression. Green triangles, H3K4me3 marks on chromatin; red circles, H3K27me3 marks.
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Igf2bp2 functions during cell differentiation and tumorigenesisWehave identified Igf2bp2 as amenin gene target that shows an
increase in H3K4me3 levels and reduction in H3K27me3, uponMen1 loss. We also observe that this inverse correlation isenhanced with increasing age and is most dramatic in Men1-deficient tumors.We found that Igf2bp2 is thefirst gene that showsan inverse correlation between H3K4me3 and H3K27me3 levelsin neuroendocrine tumor formation in vivo and thus may repre-sent a new class of menin-regulated genes associated with thispattern of dual histone modifications. Accordingly, Igf2bp2expression can be adjusted not only by a decrease in H3K4me3but also by enhancement of H3K27me3. Igf2bp2 is a develop-mental gene highly expressed during embryogenesis, and gradu-ally silenced in the adult (39, 40). It is the major Igf2 bindingprotein family member expressed in adult pancreatic islets (WL,unpublished observations). Although the precise function ofIgf2bp2 is unclear, it has been reported that Igf2bp2 interfereswith Igf2 translation by associating with the 50 end of the Igf2transcript during embryonic development (39). It is unknownwhether Igf2bp2 also functions as an inhibitor of Igf2 or othergenes in the adult. Thus, misregulation of Igf2bp2, as observed forother developmental genes, might play a role in tumorigenesis.
Dynamic epigenetic regulation of Igf2bp2 by Men1 and Rbp2under physiologic conditions
Interestingly, menin has previously been shown to bind thepromoter of the Igfbp2 (insulin-like growth factor binding protein2) and to repress Igfbp2 expression inMEFs (41, 42), in addition tothe effect on Igf2bp2 that we report here. We do not see significantchanges for either H3K4me3 or H3K27me3 levels at the Igfbp2locus in pancreatic islets harvested frommice at age of 2 months,suggesting that the mechanism for Igfbp2 expression by menin isdifferent from that for Igf2bp2 expression.
In contrast, Igf2bp2 expression is dynamically regulated byepigenetic changes driven by menin–HMT complexes, and alsomodulated by the Rbp2 histone demethylase. We observed thatIgf2bp2 expression is decreased during the hyperplasia stage inMen1-deficient pancreatic islets and is accompanied by changes inH3K4 and H3K27 histone methylation at the Igf2bp2 promoter.These effects are partially reversed by deletion of the Rbp2 histonedemethylase, implying that inactivation of Rbp2 counteracts
epigenetic changes induced by menin–HMT complexes. Oneexplanation for this phenomenon may be that Rbp2, in associ-ation with the PRC2 complex (43), binds the Igf2bp2 promoter toreduce its expression; loss of Rbp2 may relieve this repression,resulting in gene activation.
In conclusion,wehave identified several genes, notably Igf2bp2,as being regulated by menin-mediated H3K4me3 and observeepigenetic changes in these targets over time, strongly suggesting arole for these genes in pancreatic islet tumorigenesis inducedupon ablation of Men1.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: W. Lin, H. Watanabe, M. MeyersonDevelopment of methodology: W. Lin, H. Watanabe, N. KaplanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Lin, H. Watanabe, J.M. Francis, N. Kaplan,A. AgostonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Lin, H. Watanabe, J.M. Francis, N. Kaplan,C.S. Pedamallu, A.J. BassWriting, review, and/or revision of the manuscript: W. Lin, H. Watanabe,N. Kaplan, C.S. Pedamallu, A. Ramachandran, M. MeyersonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. KaplanStudy supervision: M. Meyerson
AcknowledgmentsThe authors thankmembers of theMeyerson laboratory for critical reading of
the manuscript and helpful discussions, Harvard Specialized HistopathologyServices-Longwood for histology, the BioMicro Center for library preparationand Illumina sequencing, and the Joslin Diabetes Center Core facility for isletisolation.
Grant SupportThis work was supported by an anonymous gift (to M. Meyerson).The costs of publication of this articlewere defrayed inpart by the payment of
page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received August 18, 2014; revised November 12, 2014; accepted November26, 2014; published OnlineFirst December 23, 2014.
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