men1 is a melanoma tumor suppressor that preserves genomic integrity by stimulating transcription of...

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

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?

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

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: Melanomas:

Fig. 1A

MEN1 in Melanoma

Fig. 1B

MEN1 in Melanoma

Fig. 1C

Surveyed additional samples by immunohistochemistry

MEN1 in Melanoma

Fig. 1D

Is the expression of MEN1 reduced in melanomas

compared to normal skin and benign tumors (nevi)?

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

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

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 linesColonies counted after 14 days

Fig. 1G

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

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

Fig. 2A

Does MEN1 contribute to Genomic Stability?

Does the MEN1 knockdown affect proteins known to be involved with repair of double-strand breaks?

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

Fig. 2D

HR requires RAD51 protein

Monitored RAD51 loci

Conclusion?

MEN1 Contributes to Genome Stability

Fig. 3A

WT Melanocytes vs. Melanoma cell lines (all with low MEN1 expression)

Conclusions?

MEN1 Contributes to Genome Stability

Fig. 3B

Can we reverse these phenotypes by expressing MEN1 from a plasmid?

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

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

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

MEN1 and HR vs. NHEJ

Fig. 4A

p<0.001

Plasmid-based HR measurement

MEN1 and HR vs. NHEJ

Similar HR assay, using chromosomal integrationsTwo nonfunctional neomycin-resistance genes with different mutations

Fig. 4A

p<0.001

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

MEN1 and HR vs. NHEJ

Fig. 4B

p<0.001

Plasmid-based NHEJ Assay

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

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

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

Fig. 4B

p<0.01

Chromosome-based NHEJ assay

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

MEN1 and HR vs. NHEJ

Nature Rev. Cancer 9:371

Why does MEN1 affect DSB repair?

Is MEN1 an ATM substrate?

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 assayGST-MEN1 purified from E. coliFlag-tagged ATM IP’d from murine cells32P-ATP

Fig. 5A

MEN1 and ATM

Fig. 5B

Is the effect ATM-dependent?

Conclusions?

MEN1 and ATM

Fig. 5D

Same experiment but with the mutant MEN1

What question is Fang et al. asking?

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

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

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 Significant enrichment

Three biological replicates from HeLa cells

MEN1 Location in the Human Genome

PLoS Genetics 2006 2(4):e51

(bla

ck p

oint

s)

(red

poi

nts)

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…

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

MEN1 and Transcription of Repair Genes

Fig. 6C

Normalized to no antibody control (=1)

PCR primers for indicated promoters (P) or last exon (E)

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

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

Fig. 8A

MEN1 associates with MLL

Knockdown expression and measure target gene mRNA levels by qRT-PCR

MEN1 and Transcription of Repair Genes

Does estradiol affect the transcription of these genes?

Melanocytes treated or untreatedqRT-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

Fig. 8F

Are the data consistent with the model?

MEN1 and Transcription of Repair Genes

Fig. 9A

Conclusion?

MEN1 and Transcription of Repair Genes

Fig. 9B

Does the DSB response cause these proteins to bind to the promoters?

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?

MEN1 and Transcription of Repair Genes

Fig. 9C

Estradiol and ionizing radiation synergize in the activation of HR genes

MEN1 and Transcription of Repair Genes

Fig. 9D

Is this just because we’re getting more ERa?

MEN1 and Transcription of Repair Genes

Fig. 9D

Is this just because the cells are producing their own estradiol?

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

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 A176Pall 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

Fig. 7F

What can we conclude about these MEN1 mutants?

Are they truly loss-of-function mutants?

MEN1 and Transcription of Repair Genes

Fig. 9F