men1 is a melanoma tumor suppressor that preserves genomic integrity by stimulating transcription of...
TRANSCRIPT
![Page 1: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/1.jpg)
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 SchoolOhio State University
Boston UniversityYale University
Molecular and Cellular Biology33:2635-2647
July 2013
![Page 2: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/2.jpg)
Multiple Endocrine Neoplasia type 1 (MEN1) Syndrome
Surgery 144:695
Frequent tumors in endocrine tissuesespecially pancreas, adrenal glands, pituitary and parathyroid
What can you tell about it’s inheritance?
![Page 3: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/3.jpg)
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 domainsExpressed in most tissuesIn NucleusMany binding partners have been identified
Trends Biochem. Sci, in press
![Page 4: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/4.jpg)
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?
![Page 5: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/5.jpg)
MEN1 in Melanoma
Is the expression of MEN1 reduced in melanomas compared to
normal skin and benign tumors (nevi)? Measure with qRT-PCR
Nevi: Melanomas:
Fig. 1A
![Page 6: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/6.jpg)
MEN1 in Melanoma
Fig. 1B
![Page 7: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/7.jpg)
MEN1 in Melanoma
Fig. 1C
Surveyed additional samples by immunohistochemistry
![Page 8: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/8.jpg)
MEN1 in Melanoma
Fig. 1D
Is the expression of MEN1 reduced in melanomas
compared to normal skin and benign tumors (nevi)?
![Page 9: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/9.jpg)
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
Genomic DNA is fragmented
-CH
3
-CH3
-CH
3
-CH3
-CH
3
-CH3
![Page 10: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/10.jpg)
MEN1 in Melanoma
MBD (methyl binding domain) connected to insoluble beads
Methylated DNA is precipitated.
Unmethylated DNA is washed away.
Specific DNA is detected by PCR
-CH
3
-CH3
-CH
3
-CH3
MBD MBD
![Page 11: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/11.jpg)
MEN1 in Melanoma
Fig. 1F
![Page 12: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/12.jpg)
MEN1 in Melanoma
If MEN1 is a TSG, it should suppress colony formation
MEN1 expression plasmid (or control) was trasfected into melanoma cell linesColonies counted after 14 days
Fig. 1G
![Page 13: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/13.jpg)
MEN1 Contributes to Genome Stability
Does MEN1 contribute to Genomic Stability?
MEN1 knockdown in primary human melanocytesshRNA = short hairpin RNA
Confirm knockdown by qRT-PCR and western blot
Fig. 2AB
![Page 14: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/14.jpg)
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)
![Page 15: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/15.jpg)
MEN1 Contributes to Genome Stability
Fig. 2A
Does MEN1 contribute to Genomic Stability?
Does the MEN1 knockdown affect proteins known to be involved with repair of double-strand breaks?
![Page 16: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/16.jpg)
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
![Page 17: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/17.jpg)
MEN1 Contributes to Genome Stability
Fig. 2D
HR requires RAD51 protein
Monitored RAD51 loci
Conclusion?
![Page 18: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/18.jpg)
MEN1 Contributes to Genome Stability
Fig. 3A
WT Melanocytes vs. Melanoma cell lines (all with low MEN1 expression)
Conclusions?
![Page 19: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/19.jpg)
MEN1 Contributes to Genome Stability
Fig. 3B
Can we reverse these phenotypes by expressing MEN1 from a plasmid?
![Page 20: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/20.jpg)
MEN1 and HR vs. NHEJ
Measuring HR rates in vivo
Transfect mammalian cells with two plasmidboth have AmpR genes but defective KanR genesbut different mutations in KanR genes
AmpR
Defective KanR Gene
AmpR
Defective KanR Gene
![Page 21: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/21.jpg)
MEN1 and HR vs. NHEJ
Measuring HR rates in vivo
HR can join the plasmids together, making one functional KanR gene
AmpR
Defective KanR Gene
AmpR
Defective KanR Gene
HR
AmpR
AmpR
Def
ectiv
e Ka
nR Gen
eFu
nctio
nal K
anR G
ene
![Page 22: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/22.jpg)
MEN1 and HR vs. NHEJ
AmpR
Defective KanR Gene
AmpR
Defective KanR Gene
HR
AmpR
AmpR
Def
ectiv
e Ka
nR Gen
eFu
nctio
nal K
anR G
ene
Collect DNA from cells.
Transform into E. coli. Measure fraction of AmpR cells that are also KanR
![Page 23: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/23.jpg)
MEN1 and HR vs. NHEJ
Fig. 4A
p<0.001
Plasmid-based HR measurement
![Page 24: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/24.jpg)
MEN1 and HR vs. NHEJ
Similar HR assay, using chromosomal integrationsTwo nonfunctional neomycin-resistance genes with different mutations
Fig. 4A
p<0.001
![Page 25: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/25.jpg)
MEN1 and HR vs. NHEJ
Plasmid-based assay for NHEJ
Transfect mammalian cells with two plasmids:
linear plasmid (AmpR)circular plasmid (KanR, control)
48 hr later, collect DNATransform into E. coli
AmpR DNA can only be maintained if it has been circularized
Find ratio of AmpR to KanR transformants
KanR
AmpR
NHEJ
KanR AmpR
![Page 26: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/26.jpg)
MEN1 and HR vs. NHEJ
Fig. 4B
p<0.001
Plasmid-based NHEJ Assay
![Page 27: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/27.jpg)
MEN1 and HR vs. NHEJ
Chromosome-based NHEJ assay
HEK293/pPHW1 cells have this artificial DNA integrated in the genome
GPT gene
Mini-ORFwith ATG
SceI SiteSceI Site
As is, transcription happens but GPT can’t be translated
GPT is needed for the cell to survive the drug XHATM
![Page 28: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/28.jpg)
MEN1 and HR vs. NHEJ
Chromosome-based NHEJ assay
HEK293/pPHW1 cells have this artificial DNA integrated in the genome
GPT gene
Mini-ORFwith ATG
SceI SiteSceI Site
Transfect with the gene encoding the SceI restriction enzyme
GPT gene
![Page 29: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/29.jpg)
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!
![Page 30: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/30.jpg)
MEN1 and HR vs. NHEJ
Fig. 4B
p<0.01
Chromosome-based NHEJ assay
![Page 31: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/31.jpg)
MEN1 and HR vs. NHEJ
NHEJ leads to more sequence errors than HR
Measure mutation rate by looking for loss of HPRT gene function
HPRT6-thioguanine 6-thioguanosine monophosphate
toxic
Melanocytes or HCT116 cells MEN1 knockdown look at fraction of cells that survive 6-TG
Fig. 4CD
![Page 32: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/32.jpg)
MEN1 and HR vs. NHEJ
Nature Rev. Cancer 9:371
Why does MEN1 affect DSB repair?
Is MEN1 an ATM substrate?
![Page 33: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/33.jpg)
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
![Page 34: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/34.jpg)
MEN1 and ATM
in vitro kinase assayGST-MEN1 purified from E. coliFlag-tagged ATM IP’d from murine cells32P-ATP
Fig. 5A
![Page 35: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/35.jpg)
MEN1 and ATM
Fig. 5B
Is the effect ATM-dependent?
Conclusions?
![Page 36: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/36.jpg)
MEN1 and ATM
Fig. 5D
Same experiment but with the mutant MEN1
What question is Fang et al. asking?
![Page 37: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/37.jpg)
MEN1 and ATM
Fig. 5E
Hypothesis: ATM phosphorylates MEN1 protein to prevent polyubiquitination
Same system as beforeAlso express Ubiquitin tagged with HA epitope
IP with anti-HA antibody
Western blot with anti-MEN1 antibody
![Page 38: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/38.jpg)
MEN1 and Transcription of Repair Genes
MEN1 is known to bind to many transcription factorsreported 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
![Page 39: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/39.jpg)
MEN1 Location in the Human Genome
Cy5 label ChIP’d DNA
Cy3 label total DNA
![Page 40: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/40.jpg)
MEN1 Location in the Human Genome
PLoS Genetics 2006 2(4):e51
log2 Significant enrichment
Three biological replicates from HeLa cells
![Page 41: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/41.jpg)
MEN1 Location in the Human Genome
PLoS Genetics 2006 2(4):e51
(bla
ck p
oint
s)
(red
poi
nts)
![Page 42: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/42.jpg)
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…
![Page 43: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/43.jpg)
MEN1 and Transcription of Repair Genes
MEN1 is known to bind to many transcription factorsreported 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
![Page 44: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/44.jpg)
MEN1 and Transcription of Repair Genes
Fig. 6C
Normalized to no antibody control (=1)
PCR primers for indicated promoters (P) or last exon (E)
![Page 45: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/45.jpg)
MEN1 and Transcription of Repair Genes
Fig. 8A
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
![Page 46: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/46.jpg)
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
![Page 47: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/47.jpg)
MEN1 and Transcription of Repair Genes
Fig. 8A
MEN1 associates with MLL
Knockdown expression and measure target gene mRNA levels by qRT-PCR
![Page 48: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/48.jpg)
MEN1 and Transcription of Repair Genes
Does estradiol affect the transcription of these genes?
Melanocytes treated or untreatedqRT-PCR
Fig. 8B
![Page 49: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/49.jpg)
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
![Page 50: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/50.jpg)
MEN1 and Transcription of Repair Genes
Does fulvestrant (an anti-estrogen) affect these genes?
Fig. 8C
![Page 51: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/51.jpg)
MEN1 and Transcription of Repair Genes
Diminished ESR1 activity reduces HR and increases NHEJ – just like loss of MEN1 function
Fig. 8E
![Page 52: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/52.jpg)
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
![Page 53: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/53.jpg)
MEN1 and Transcription of Repair Genes
Who binds to whom?
Test with knockdowns and ChIPs
Fig. 8F
Are the data consistent with the model?
![Page 54: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/54.jpg)
MEN1 and Transcription of Repair Genes
Fig. 9A
Conclusion?
![Page 55: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/55.jpg)
MEN1 and Transcription of Repair Genes
Fig. 9B
Does the DSB response cause these proteins to bind to the promoters?
![Page 56: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/56.jpg)
MEN1 and Transcription of Repair Genes
Fig. 8B, 9A
Estradiol induces HR gene expression
IR induces HR gene expression
What if we put them together?
![Page 57: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/57.jpg)
MEN1 and Transcription of Repair Genes
Fig. 9C
Estradiol and ionizing radiation synergize in the activation of HR genes
![Page 58: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/58.jpg)
MEN1 and Transcription of Repair Genes
Fig. 9D
Is this just because we’re getting more ERa?
![Page 59: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/59.jpg)
MEN1 and Transcription of Repair Genes
Fig. 9D
Is this just because the cells are producing their own estradiol?
![Page 60: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/60.jpg)
MEN1 and Transcription of Repair Genes
Many melanomas have reduced transcription of MEN1
Do they also have reduced expressionof BRCA1?
p21 as a positive control (known to be a MEN1 target gene,ref. 8)
Fig. 1A
![Page 61: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/61.jpg)
MEN1 and Transcription of Repair Genes
Many melanomas have reduced transcription of MEN1
Do they also have reduced expression of BRCA1?
Fig. 10A
![Page 62: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/62.jpg)
MEN1 Mutations
MEN1 gene was first identified in familial multiple endocrine neoplasia
Commonly occurring MEN1 mutations are H139D, A160P and A176Pall are loss-of-function mutations
Do they affect these phenotypes?
Overexpress the mutants in melanocytes
Fig. 7A
![Page 63: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/63.jpg)
MEN1 Mutations
Are the mutant MEN1 proteins stabilized by ATM phosphorylation?
Fig. 7B
![Page 64: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/64.jpg)
MEN1 Mutations
What question are Fang et al. asking with this experiment?
Fig. 7C
![Page 65: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/65.jpg)
MEN1 Mutations
What question are Fang et al. asking with this experiment?
Fig. 7D
![Page 66: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/66.jpg)
MEN1 Mutations
What question are Fang et al. asking with this experiment?
Fig. 7E
![Page 67: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/67.jpg)
MEN1 Mutations
What question are Fang et al. asking with this experiment?
Fig. 7F
![Page 68: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/68.jpg)
MEN1 Mutations
Fig. 7F
What can we conclude about these MEN1 mutants?
Are they truly loss-of-function mutants?
![Page 69: MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed](https://reader035.vdocuments.mx/reader035/viewer/2022062421/56649d965503460f94a7f6cf/html5/thumbnails/69.jpg)
MEN1 and Transcription of Repair Genes
Fig. 9F