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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

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