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Dynamic Epigenetic Regulation by Menin During Pancreatic Islet Tumor Formation Wenchu Lin1,2,3,4, Hideo Watanabe1,2,3, Shouyong Peng1,2,3, Joshua M

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Dynamic Epigenetic Regulation by Menin During Pancreatic Islet Tumor Formation Wenchu Lin1,2,3,4, Hideo Watanabe1,2,3, Shouyong Peng1,2,3, Joshua M Published OnlineFirst December 23, 2014; DOI: 10.1158/1541-7786.MCR-14-0457 Chromatin, Gene, and RNA Regulation Molecular Cancer Research Dynamic Epigenetic Regulation by Menin During Pancreatic Islet Tumor Formation Wenchu 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 in with a concomitant decrease in H3K4me3 within the promoters sporadic pancreatic neuroendocrine tumors (PanNET) and is of these target genes. In particular, expression of the insulin-like responsible for the familial multiple endocrine neoplasia type 1 growth factor 2 mRNA binding protein 2 (IGF2BP2)geneis (MEN-1) cancer syndrome. Menin, the protein product of subject to dynamic epigenetic regulation by Men1-dependent MEN1, associates with the histone methyltransferases (HMT) histone modification in a time-dependent manner. Decreased MLL1 (KMT2A) and MLL4 (KMT2B) to form menin–HMT expression of IGF2BP2 in Men1-deficient hyperplastic pancre- complexes in both human and mouse model systems. To atic islets is partially reversed by ablation of RBP2 (KDM5A), a elucidate the role of methylation of histone H3 at lysine 4 histone H3K4-specific demethylase of the jumonji, AT-rich (H3K4) mediated by menin–HMT complexes during PanNET interactive domain 1 (JARID1) family. Taken together, these formation, genome-wide histone H3 lysine 4 trimethylation data demonstrate that loss of Men1 in pancreatic islet cells alters (H3K4me3) signals were mapped in pancreatic islets using the epigenetic landscape of its target genes. unbiased chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Integrative analysis of Implications: Epigenetic profiling and gene expression analysis in gene expression profiles and histone H3K4me3 levels identified Men1-deficient pancreatic islet cells reveals vital insight into the a number of transcripts and target genes dependent on menin. molecular events that occur during the progression of pancreatic In the absence of Men1, histone H3K27me3 levels are enriched, islet tumorigenesis. Mol Cancer Res; 13(4); 689–98. Ó2014 AACR. Introduction recapitulating the spectrum of tumors in MEN-1 syndrome, with Men1 conditional knockout animals demonstrating a Multiple endocrine neoplasia type 1 (MEN-1) is an auto- shorter latency (6–9). Although Men1 mutations are primarily somal-dominant syndrome, characterized by multiple tumors associated with neuroendocrine cancers, several lines of evi- in endocrine tissues such as the pituitary gland, parathyroid dence demonstrate that Men1 can also be dysregulated in gland, and pancreatic islets (1). Linkage studies and posi- non-neuroendocrine tumors such as lung cancer, melanoma, tional cloning identified the causative gene, MEN1,forthis and liver cancer (10–12). disorder. Over 1,300 mutations, typically truncating, have Several studies have implicated that menin, the protein product been identified in MEN1 (2, 3). The importance of MEN1 of MEN1, is involved in transcriptional regulation, cell-cycle inactivation in tumorigenesis is highlighted by the frequency control, protein degradation, and genome instability through of MEN1 mutations in sporadic endocrine tumors—44% in interaction with a number of transcription factors such as JunD, pancreatic neuroendocrine tumors and 35% in parathyroid NF-kB, and members of the Smad family (3, 13–15). In addition, adenomas (4, 5). Heterozygous and conditional Men1 knock- we and others have shown that menin is physically associated out mice develop tumors in multiple neuroendocrine tissues, 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 1 Department of Medical Oncology, Center for Cancer Genome Dis- (H3K4me3; refs. 16, 17). Surprisingly, menin also binds to the covery, Dana-Farber Cancer Institute, Boston, Massachusetts. 2Department of Pathology, Brigham and Women's Hospital and MLL fusion protein in leukemia cells to upregulate HoxA9 gene Harvard Medical School, Boston, Massachusetts. 3Cancer program, expression, thus promoting oncogenic activity in MLL-associated Broad Institute of Harvard and MIT, Cambridge, Massachusetts. leukemiogenesis (18). Genome-wide analysis by chromatin 4High Magnetic Field Laboratory, Chinese Academy of Sciences, 350 Shushanhu RD, Hefei, Anhui Province, 230031, P. R. China. immunoprecipitation coupled with DNA microarray analysis (ChIP-chip; ref. 19) has revealed that menin colocalizes with Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). MLL at gene promoters in various cell types, suggesting that menin regulates transcription in cooperation with MLL in multiple W. Lin and H. Watanabe contributed equally to this article. tissues (20). Corresponding Author: Matthew Meyerson, Dana-Farber Cancer Institute, Currently, there are no targeted therapies directed toward Boston, MA 02215. Phone: 617-632-4768; Fax: 617-582-7880; E-mail: patients harboring pancreatic neuroendocrine cancer tumors with [email protected] MEN1 mutations. Thus, there is a critical need to deepen our doi: 10.1158/1541-7786.MCR-14-0457 understanding of the biology of these cancers to develop more Ó2014 American Association for Cancer Research. effective therapeutic approaches. Given the inactivation of menin www.aacrjournals.org 689 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 23, 2014; DOI: 10.1158/1541-7786.MCR-14-0457 Lin et al. in multiple endocrine cancers and the reversibility of histone Histologic and immunohistochemical analysis for pancreatic H3K4me3, we were interested in the role of enzymes that poten- tissues tially antagonize the histone methylation activity of menin. Rbp2 Pancreata were collected from mice at indicated time points (Kdm5a, Jarid1a), initially identified as Retinoblastoma-binding and fixed in 4% paraformaldehyde for 2 hours followed by protein 2, is a member of the Jumonji (JMJ) domain-containing dehydration and paraffin embedding. Histopathologic analysis family of histone demethylases, with roles in chromatin modi- was carried out on 5 mm sections stained with hematoxylin and fication and transcriptional regulation (21). Loss of Rbp2 recruit- eosin. Islet morphology and tumors were examined in at least ment to the CDKN1B gene is highly correlated with increased three cut sections for each pancreas after staining with hematox- histone H3K4me3 levels and elevated gene expression (21–23). ylin and eosin. Appropriate positive and negative controls were We have previously demonstrated in murine models that inacti- run on matched sections for all applied antibodies. Immunohis- vation of the histone demethylase Rbp2 significantly inhibits tochemical staining was performed on serial sections using anti- tumor growth in Men1-deficent mice (24). We also demonstrated bodies against H3K4me3 (Active Motif, catalog no. 36159, that alterations in gene expression patterns upon Men1 loss in 1:500), H3K27me3 (Cell Signaling Technology, catalog no. pancreatic islets are partially reversed by Rbp2 loss in these cells 9733, 1:100) and Igf2bp2 (Abcam, Ab124930, 1:1,000). Sections (24). Collectively, these observations support the notion that (i) were counterstained in Meyer hematoxylin, mounted, and photo- histone methylation plays a key role in Men1 deletion-mediated graphed using an Olympus microscope. tumorigenesis in neuroendocrine cells, and (ii) the demethylase enzyme activity of Rbp2 antagonizes the histone methyltransfer- ChIP-seq ase activity associated with menin at gene loci such as CDKN1B. For each ChIP experiment, islets from at least 4 adult mice Although loss-of-function of menin is known to play an impor- were purified by collagenase digestion and gradient centrifuga- tant role in tumor initiation and progression in endocrine tissues tion, with subsequent hand picking. ChIP was performed as (7), there is limited information on the mechanisms linking described (26) using 4 mg of anti-H3K4me3 (Active Motif, catalog – fi menin HMT complexes to neuroendocrine-speci c hyperplasia no. 36159) or anti-H3K27me3 antibodies (Cell Signaling Tech- and tumorigenesis. nology, catalog no. 9733). Five to 50 ng of DNA was used for fi Men1-de cient mice can take up to a year to accumulate the library construction. DNA was prepared for sequencing by Illu- numerous genetic and epigenetic alterations that result in tumor mina cluster generation using a SPRI-work system with 100–300 formation, a slow process during which pancreatic islet cells bp size selection followed by enrichment with barcoded PCR fi transform from normal to a hyperplastic and nally a malignant primers for multiplexing. Sequencing was performed on a state (7). Thus, this period represents a window of opportunity to Hiseq2000 machine for 40 nucleotides from a single end, at the investigate early events leading to tumorigenesis and to address MIT BioMicro Center. Barcode-separated FASTQ files were gen- – the role of menin HMT complexes in modulating cell prolifer- erated from QSEQ files. ation and behavior at this precancerous stage. To investigate tumor formation mediated by alterations in ChIP-Seq data analysis H3K4me3 levels and to identify gene targets of menin–HMT
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