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MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed DNA Repair Minggang Fang, Fen Xia, Meera Mahalingam, Ching-Man Virbasius, Narendra Wajapeyee and Michael R. Green University of Massachusetts Medical School Ohio State University Boston University Yale University Molecular and Cellular Biology 33:2635-2647 July 2013 Multiple Endocrine Neoplasia type 1 (MEN1) Syndrome Frequent tumors in endocrine tissues especially pancreas, adrenal glands, pituitary and parathyroid What can you tell about it’s inheritance? Surgery 144:695 Multiple Endocrine Neoplasia type 1 (MEN1) Syndrome Caused by loss-of-function mutations in MEN1 gene (also known as menin) But what does this protein normally do? No clear domains Expressed in most tissues In Nucleus Many binding partners have been identified Trends Biochem. Sci, in press Multiple Endocrine Neoplasia type 1 (MEN1) Syndrome MEN1-/- tumors show increased incidence of chromosome breakage and instability MEN1 has been implicated in non-endocrine tumors including lung, breast, prostate and skin cancers Michael Green’s lab has shown that MEN1 is needed for oncogenic BRAF to induce senescence in melanocytes (Cell132:363) Melanoma tumor suppressor? MEN1 in Melanoma Is the expression of MEN1 reduced in melanomas compared to normal skin and benign tumors (nevi)? Measure with qRT-PCR Nevi: Fig. 1A Melanomas: MEN1 in Melanoma Fig. 1B MEN1 in Melanoma Surveyed additional samples by immunohistochemistry Fig. 1C MEN1 in Melanoma Is the expression of MEN1 reduced in melanomas compared to normal skin and benign tumors (nevi)? Fig. 1D MEN1 in Melanoma Is the MEN1 gene not expressed due to methylation? Different assay than the bisulfite that Zhang et al. used. Similar to ChIP -CH3 Genomic DNA is fragmented -CH3 -CH3 -CH3 -CH3 -CH3 MEN1 in Melanoma MBD (methyl binding domain) connected to insoluble beads Methylated DNA is precipitated. Unmethylated DNA is washed away. -CH3 -CH3 Specific DNA is detected by PCR -CH3 -CH3 MBD MBD MEN1 in Melanoma Fig. 1F MEN1 in Melanoma If MEN1 is a TSG, it should suppress colony formation MEN1 expression plasmid (or control) was trasfected into melanoma cell lines Colonies counted after 14 days Fig. 1G MEN1 Contributes to Genome Stability Does MEN1 contribute to Genomic Stability? MEN1 knockdown in primary human melanocytes shRNA = short hairpin RNA Confirm knockdown by qRT-PCR and western blot Fig. 2AB MEN1 Contributes to Genome Stability Many types of DNA damage and DNA repair. Focus on double-strand breaks (DSBs) Alternative histone H2AX is recruited to DSBs Repaired by either Homologous Recombination (HR) or Nonhomologous End Joining (NHEJ) MEN1 Contributes to Genome Stability Does MEN1 contribute to Genomic Stability? Does the MEN1 knockdown affect proteins known to be involved with repair of doublestrand breaks? Fig. 2A MEN1 Contributes to Genome Stability Determine the location of H2AX by Immunofluorescence (IF) Permeablized cells on a microscope slide Primary antibody that binds H2AX Secondary antibody that is fluorescent Stain nuclear DNA with DAPI MEN1 Contributes to Genome Stability HR requires RAD51 protein Monitored RAD51 loci Conclusion? Fig. 2D MEN1 Contributes to Genome Stability WT Melanocytes vs. Melanoma cell lines (all with low MEN1 expression) Conclusions? Fig. 3A MEN1 Contributes to Genome Stability Can we reverse these phenotypes by expressing MEN1 from a plasmid? Fig. 3B MEN1 and HR vs. NHEJ Measuring HR rates in vivo Transfect mammalian cells with two plasmid both have AmpR genes but defective KanR genes but different mutations in KanR genes AmpR AmpR Defective KanR Gene Defective KanR Gene MEN1 and HR vs. NHEJ Measuring HR rates in vivo HR can join the plasmids together, making one functional KanR gene HR Defective KanR Gene Defective KanR Gene Functional KanR Gene AmpR AmpR AmpR Defective KanR Gene AmpR MEN1 and HR vs. NHEJ Collect DNA from cells. Transform into E. coli. Measure fraction of AmpR cells that are also KanR HR Defective KanR Gene Defective KanR Gene Functional KanR Gene AmpR AmpR AmpR Defective KanR Gene AmpR MEN1 and HR vs. NHEJ Plasmid-based HR measurement p<0.001 Fig. 4A MEN1 and HR vs. NHEJ Similar HR assay, using chromosomal integrations Two nonfunctional neomycin-resistance genes with different mutations p<0.001 Fig. 4A MEN1 and HR vs. NHEJ KanR Plasmid-based assay for NHEJ Transfect mammalian cells with two plasmids: linear plasmid (AmpR) circular plasmid (KanR, control) AmpR 48 hr later, collect DNA Transform into E. coli AmpR DNA can only be maintained if it has been circularized Find ratio of AmpR to KanR transformants NHEJ KanR AmpR MEN1 and HR vs. NHEJ Plasmid-based NHEJ Assay p<0.001 Fig. 4B MEN1 and HR vs. NHEJ Chromosome-based NHEJ assay HEK293/pPHW1 cells have this artificial DNA integrated in the genome SceI Site SceI Site GPT gene Mini-ORF with ATG As is, transcription happens but GPT can’t be translated GPT is needed for the cell to survive the drug XHATM MEN1 and HR vs. NHEJ Chromosome-based NHEJ assay HEK293/pPHW1 cells have this artificial DNA integrated in the genome SceI Site SceI Site GPT gene Mini-ORF with ATG Transfect with the gene encoding the SceI restriction enzyme GPT gene MEN1 and HR vs. NHEJ Chromosome-based NHEJ assay HEK293/pPHW1 cells have this artificial DNA integrated in the genome GPT gene NHEJ GPT gene Cells survive XHATM! MEN1 and HR vs. NHEJ Chromosome-based NHEJ assay p<0.01 Fig. 4B MEN1 and HR vs. NHEJ NHEJ leads to more sequence errors than HR Measure mutation rate by looking for loss of HPRT gene function HPRT 6-thioguanine 6-thioguanosine monophosphate toxic Melanocytes or HCT116 cells MEN1 knockdown look at fraction of cells that survive 6-TG Fig. 4CD MEN1 and HR vs. NHEJ Why does MEN1 affect DSB repair? Is MEN1 an ATM substrate? Nature Rev. Cancer 9:371 MEN1 and ATM MEN1 is one of many proteins reported to be phosphorylated by ATM (ref. 34, 35) ATM phosphorylates Ser/Thr amino acids followed by Gln (Q) Which amino acid(s) of MEN1 is phosphorylated by ATM? Ser394 and Ser399? Science 316:1160 Fig. 5A MEN1 and ATM in vitro kinase assay GST-MEN1 purified from E. coli Flag-tagged ATM IP’d from murine cells 32P-ATP Fig. 5A MEN1 and ATM Is the effect ATM-dependent? Conclusions? Fig. 5B MEN1 and ATM Same experiment but with the mutant MEN1 What question is Fang et al. asking? Fig. 5D MEN1 and ATM Hypothesis: ATM phosphorylates MEN1 protein to prevent polyubiquitination Same system as before Also express Ubiquitin tagged with HA epitope IP with anti-HA antibody Western blot with anti-MEN1 antibody Fig. 5E MEN1 and Transcription of Repair Genes MEN1 is known to bind to many transcription factors reported to be present at ~2,000 promoters in human cells Does MEN1 affect the transcription of repair genes? qRT-PCR of selected genes. Which are controls (positive and negative)? Fig. 6A MEN1 Location in the Human Genome Cy5 label ChIP’d DNA Cy3 label total DNA MEN1 Location in the Human Genome PLoS Genetics 2006 2(4):e51 log2 Three biological replicates from HeLa cells Significant enrichment (black points) (red points) MEN1 Location in the Human Genome PLoS Genetics 2006 2(4):e51 MEN1 Location in the Human Genome random segment of chromosome 3 shows four putative MEN1 binding sites zooming in… PLoS Genetics 2006 2(4):e51 MEN1 and Transcription of Repair Genes MEN1 is known to bind to many transcription factors reported to be present at ~2,000 promoters in human cells Does MEN1 affect the transcription of repair genes? qRT-PCR of selected genes Fig. 6A MEN1 and Transcription of Repair Genes Normalized to no antibody control (=1) PCR primers for indicated promoters (P) or last exon (E) Fig. 6C MEN1 and Transcription of Repair Genes MEN1 isn’t a DNA binding protein. How is it getting to promoters? By binding to estrogen receptor a (ESR1)? Knockdown expression and measure target gene mRNA levels by qRT-PCR Fig. 8A MEN1 and Transcription of Repair Genes MEN1 associates with MLL a H3K4 methyltransferase – coactivator protein MLL translocations are associated with ~10% of leukemias Bioessays 34:771 MEN1 and Transcription of Repair Genes MEN1 associates with MLL Knockdown expression and measure target gene mRNA levels by qRT-PCR Fig. 8A MEN1 and Transcription of Repair Genes Does estradiol affect the transcription of these genes? Melanocytes treated or untreated qRT-PCR Fig. 8B MEN1 and Transcription of Repair Genes Does estradiol affect which proteins are at these promoters? Melanocytes treated or untreated. ChIP, normalized to no-antibody control Fig. 8C MEN1 and Transcription of Repair Genes Does fulvestrant (an anti-estrogen) affect these genes? Fig. 8C MEN1 and Transcription of Repair Genes Diminished ESR1 activity reduces HR and increases NHEJ – just like loss of MEN1 function Fig. 8E MEN1 and Transcription of Repair Genes Who binds to whom? Expectations: Thus, MEN1 binding to the promoter should depend on ESR1 but not on MLL MLL binding should depend on ESR1 and MEN1 Histone methylation should depend on ESR1, MEN1 and MLL Fig. 9F MEN1 and Transcription of Repair Genes Who binds to whom? Test with knockdowns and ChIPs Are the data consistent with the model? Fig. 8F MEN1 and Transcription of Repair Genes Conclusion? Fig. 9A MEN1 and Transcription of Repair Genes Does the DSB response cause these proteins to bind to the promoters? Fig. 9B MEN1 and Transcription of Repair Genes Estradiol induces HR gene expression IR induces HR gene expression What if we put them together? Fig. 8B, 9A MEN1 and Transcription of Repair Genes Estradiol and ionizing radiation synergize in the activation of HR genes Fig. 9C MEN1 and Transcription of Repair Genes Is this just because we’re getting more ERa? Fig. 9D MEN1 and Transcription of Repair Genes Is this just because the cells are producing their own estradiol? Fig. 9D MEN1 and Transcription of Repair Genes Many melanomas have reduced transcription of MEN1 Do they also have reduced expression of BRCA1? p21 as a positive control (known to be a MEN1 target gene, ref. 8) Fig. 1A MEN1 and Transcription of Repair Genes Many melanomas have reduced transcription of MEN1 Do they also have reduced expression of BRCA1? Fig. 10A MEN1 Mutations MEN1 gene was first identified in familial multiple endocrine neoplasia Commonly occurring MEN1 mutations are H139D, A160P and A176P all are loss-of-function mutations Do they affect these phenotypes? Overexpress the mutants in melanocytes Fig. 7A MEN1 Mutations Are the mutant MEN1 proteins stabilized by ATM phosphorylation? Fig. 7B MEN1 Mutations What question are Fang et al. asking with this experiment? Fig. 7C MEN1 Mutations What question are Fang et al. asking with this experiment? Fig. 7D MEN1 Mutations What question are Fang et al. asking with this experiment? Fig. 7E MEN1 Mutations What question are Fang et al. asking with this experiment? Fig. 7F MEN1 Mutations What can we conclude about these MEN1 mutants? Are they truly loss-of-function mutants? Fig. 7F MEN1 and Transcription of Repair Genes Fig. 9F