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Gehana Vaswani
ROLE OF MOF IN ATM SIGNALING CASCADE AND TUMORIGENESIS
BACKGROUND INFORMATION:
The DNA damage response
The cell response to DNA damage is a critical step for keeping the genome intact. Cells are
constantly undergoing DNA damage due to exposure to endogenous and exogenous damaging
agents, such as chemicals, ionizing irradiation (IR), ultra violet rays (UV), therapeutic agents,
and the products of normal metabolism. Damages to DNA affect modification and degradation of
proteins, chromatin remodeling, gene transcription, and transport of macromolecules. Cell
exposure to IR is an excellent model for studying DNA damage. In fact, IR induces the
production of double-strand breaks (DSBs), which are the most cytotoxic DNA lesions. One of
the first molecules involved in the response of IR-induced DNA damage is the ATM protein.
The ATM (Ataxia Telangiectasia Mutated) protein
ATM is a nuclear protein kinase with a catalytic domain similar to that of the
phosphatidylinositol 3-kinases (PI 3-kinases). ATM was discovered as an altered protein in
patients with ataxia-telagiectasia (A-T), a severe genetic disorder characterized by cerebellar
degeneration, neuromotor dysfunction, chromosomal instability, immune system defects, cancer
predisposition, and acute sensitivity to ionizing radiation. In undamaged cells, ATM is present as
a dimer or oligomer in which the kinase domain is silent because it is associated with the FAT
region of another ATM monomer (Gupta et al. 2005). Following DSB formation, ATM rapidly
autophosphorylates serine 1981, and the inactive ATM dimers are dissociated into active ATM
monomers. Active phosphorylated ATM molecules phosphorylate downstream proteins that
affect one or more cell cycle checkpoints. Some of the known substrates are the p53 protein and
its ubiquitin ligase; MDM2; the Nbs1 protein; the BRCA1 protein, which interacts with other
repair proteins; the checkpoint kinase 2, Chk2; the Rad17 protein; and the chromatin remodeling
protein SMC1. Because of the involvement of ATM in these multiple pathways, the
phosphorylation of a single substrate does not provide an accurate picture of the ATM-mediated
cellular response to DNA damage. Moreover, a comparison between the signaling pathway of
ATM and those of other PI-3-like kinase domain proteins - ATR and DNA-PK - has shown no
clear differentiation between the signals. Several downstream steps are shared by these
molecules, suggesting a common underlying mechanism for repairing damaged DNA.
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Several mechanisms have been proposed for the activation of ATM by DNA damage: a) direct
activation through interaction with damaged DNA, b) indirect activation through interaction with
DNA repair or maintenance proteins, or c) a combination of both. Existing experimental data
support the idea that they are activated both through interactions with DNA and members of the
repair complexes (Hwang et al. 2002). For example, pre-treatment of DNA-cellulose matrix with
IR or restriction enzymes stimulates ATM binding, suggesting that ATM binds to DNA ends. In
addition, ATM is part of a super protein complex called the BRCA1-associated genome
surveillance complex (BASC), which is involved in the recognition and repair of aberrant DNA
structures. It has been found this complex contains several other proteins such as breast cancer
gene 1 (BRCA1), mismatch-repair protein hRad50, and BLM helicase (Hwang et al. 2002).
ATM also binds to histone deacetylase HDAC1 both in vitro and in vivo, and the extent of this
association was increased after exposure of MRC5CV1 human fibroblasts to IR. All these data
support the model that multiple protein complexes localize at the sites of DNA damage and
interact to trigger the checkpoint-signaling cascade.
Males absent on the first (Mof)
Histone modifications such as acetylation, methylation and phosphorylation have been
implicated in fundamental cellular processes such as epigenetic regulation of gene expression,
organization of chromatin structure, chromosome segregation, DNA replication and DNA repair.
Males absent on the first (MOF) is responsible for acetylating histone H4 at lysine 16 (H4K16)
and is a key component of the MSL complex required for dosage compensation in Drosophila.
The human ortholog of MOF (hMOF) has the same substrate specificity and recent purification
of the human and Drosophila MOF complexes showed that these complexes were also highly
conserved through evolution (Rea et al. 2007). Several studies have shown that loss of hMOF in
mammalian cells leads to a number of different phenotypes; a G2/M cell cycle arrest, nuclear
morphological defects, spontaneous chromosomal aberrations, reduced transcription of certain
genes and an impaired DNA repair response upon ionizing irradiation (Gupta et al. 2005; Rea et
al. 2007).
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The potential roles of the human ortholog of MOF (hMOF) in the cell (Gupta et al. 2008).
Acetylation of histone H4 at K16 by hMOF could:
(A) Facilitate a decrease in either the affinity of histones for DNA or in inter-nucleosomal
interactions thus opening up the chromatin structure.
(B) Act as a binding site for specific proteins that can further modify chromatin, such as
transcriptional coactivators or DNA repair proteins.
(C) Inhibit binding of specific factors such as transcriptional repressive complexes.
(D) Could result in the modification of a protein(s) important for nuclear stability. This target
could be either nuclear pore or nuclear membrane associated proteins. Following DNA damage,
hMOF may activate ATM
(E) Impact one of the cell's main repair pathways. The mechanism for this activation is unknown
but may involve increased acetylation around the sites of damage and/or acetylation of other
substrates. The tumor suppressor p53 is acetylated by hMOF
(F) Result in a decision for programmed cell death as opposed to cell cycle arrest.
Moreover, hMOF is involved in ATM activation in response to DNA damage, and acetylation of
p53 by hMOF influences the cell's decision to undergo apoptosis instead of a cell cycle arrest.
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PROPOSAL QUESTION/HYPOTHESIS:
“Mof is essential in ATM-associated DNA Damage Repair but its overexpression leads to
tumorigenesis.”
Specific Aims:
1. Investigation of proteins involved in ATM activation signaling cascade
2. Role of MOF in tumor cell growth and tumorigenesis
EXPERIMENTAL APPROACH AND EXPECTED RESULTS:
Methods used for the two proposed experiments:
Creating Mof overexpressed cells:
Plasmid construction: Plasmids for transfection will be constructed in a pIND (Invitrogen)
backbone using pBFT4 as an intermediate cloning vector. Complementary DNA for mouse Mof,
which has the same amino acid sequence as human Mof will be generated by polymerase chain
reaction (PCR). The resulting product will be incorporated into the vector adjacent to a strong
promoter for Mof, and transfected cells will be selected on Kanamycin plates.
Generation of stable transfected cell lines: Breast Cancer cell lines (MCF-7 ) (Invitrogen) will be
transfected using DMRIE-C (Life Technologies) following manufacturer's instructions in the
presence of Neomycin.
Creating Mof knockouts:
RNA Interference
Breast cancer cell lines (MCF-7) will be transfected with Mof shRNA plasmids using
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
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PROPOSED EXPERIMENTS:
PART I
Proposed experiments to establish the link between Mof and proteins involved in ATM
activation signaling cascade
To investigate proteins that link Mof and ATM activation, cells that overexpress Mof and Mof
knockout cells will be created and subjected to IR radiation. Two-dimensional polyacrylamide
gel electrophoresis can be used to purify and characterize the expressed proteins in both cell
types. As we want to find the link of Mof with the ATM pathway, we will focus on the presence
or absence of ATM downstream and upstream effectors such as Chk, BRCA 2 and other known
molecules (Ref. 6: List of known proteins in the Mof complex). We will check for their
expression in the presence and absence of Mof.
Once protein spots of interest have been revealed from the quantitative analysis of the 2-D
PAGE patterns, the problem of identification will be resolved by immunoblotting and peptidemass fingerprinting. Then we can find compare the spots to 2-D PAGE databases, there are a
number of protein spots that are already "identified" in a few cell lines. Combined with the aims
of the experiment, these databases may give one the opportunity to guess at the identity of a
particular protein spot and confirm or deny this by immunoblotting. The approach of obtaining
accurate peptide masses from specifically cleaved proteins to search protein sequence databases,
known as peptide mass fingerprinting, provides one with another opportunity to identify a
previously sequenced protein or (hopefully) confirm that it is indeed novel.
Possible outcomes:
We will identify how Mof impacts the changes in expression of proteins involved in the ATM
pathway following irradiation. The results should enable us to propose a pathway through which
DNA repair takes place after activation of ATM in the presence of Mof. In the absence of Mof,
we predict that the ATM cascade would not be activated
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PART II
Literature studies show that, the overexpression of wild-type hMOF yields a modest increase in
cell survival and enhanced DNA repair after IR exposure (Rea et al. 2007). These results suggest
that hMOF influences the function of ATM. Also, since overexpression of Mof helps cells to
avoid apoptosis, Mof may be involved in tumorigenesis.
Proposed experiments to examine the role of Mof in tumorigenesis:
To study the role of Mof in tumor formation, we will inject breast cancer cell lines (MCF-7),
which overexpress Mof or do not express Mof (knockout cells) into the mammary glands of
female CD1-NU immunodeficient (nude) mice to observe tumor growth. Before the injection of
the cells, a pellet of 17 -estradiol (0.72 mg/pellet, 60 days release time) will be implanted under
the skin in the back of the mice. Treatment with tamoxifen will be applied by replacing the
estrogen pellet with a tamoxifen pellet (5 mg/pellet, 21 days release time). The tumors will then
be investigated 4-6 wk after cell implantation.
Cell growth assay of MCF-7, MCF-7 with overexpressed Mof and Mof knockout MCF-7
cells.
All cell types will be exposed to DNA damaging agent such as IR (ionizing radiation), and then
plated in a tissue culture vessel and allowed to grow. We would predict that the cells with Mof
survive as ATM would be active in them and the repair pathway would be active. But, the Mof
knockout cells would die.
Anchorage-independent growth
Assays for anchorage-independent growth will be performed for all three types of cells to check
the tumorigenic potential of cells. Each experiment will be repeated independently three times in
duplicate, and the results will be expressed as the means of the three experiments.
Possible outcomes:
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In case of role of Mof in tumorigenesis, we predict more tumor growth in Mof overexpressed
cell lines compared to breast cancer cells. Minimal or no tumor growth is expected in Mof
knockout cells.
CONCLUSION:
These in vitro studies will determine ATM dependent and Mof functions in response to IR. We
would also be able to find the functional links among Mof, H4-K16 acetylation and IR induced
tumor formation.
MOF deficiency is involved in genomic instability and defective DNA damage repair. By this
experiment, we will whether increased amounts of Mof expression result in cells that proliferate
faster and show telltale signs of cancerous transformation. When we inject the same cells into
mice, tumors from cells that have an overabundance of Mof are predicted to grow faster than
other tumor cells. If we knockout Mof in tumor cells, we predict that they will be weakened and
unable to recover after radiation exposure.
References:
1) Gupta, T., G. Guerin-Peyrou, et al. 2008. The mammalian ortholog of Drosophila MOF
that acetylates histone H4 lysine 16 is essential for embryogenesis and oncogenesis. Mol.
Cell. Biol., 28: 397 - 409.
2) Hwang K.K. and H. J. Worman. 2002. Gene regulation by human orthologs of
Drosophila heterochromatin protein 1. Biochemical and Biophysical Research
Communications 293(4):1217-1222, DOI: 10.1016/S0006-291X(02)00377-7.
3) Sharma G.G., K.K. Hwang, et al. 2003. Human Heterochromatin Protein 1 Isoforms
HP1Hs and HP1Hsß Interfere with hTERT-Telomere Interactions and Correlate with
Changes in Cell Growth and Response to Ionizing Radiation. Molecular and Cellular
Biology, 23(22):8363-8376, DOI: 10.1128/MCB.23.22.8363-8376.2003.
4) Thomas T, Loveland KL, Voss AK. 2007. The genes coding for the MYST family
histone acetyltransferases, Tip60 and Mof, are expressed at high levels during sperm
development. Gene Expression Patterns 7(6):657-65. Epub 2007 Mar 31.
5) Gupta, A, G. G. Sharma, C.S.H. Young, M. Agarwal, E.R. Smith, T.T. Paul, J.C.
Lucchesi, K.K. Khanna, T. Ludwig, T.J. Pandita. 2005. Involvement of human MOF in
ATM function. Mol Cell Biol. 25(12):5292-305.
6) Rea S., G Xouri and A Akhtar. 2007. Males absent on the first (MOF): from flies to
humans. Oncogene 26: 5385–5394; doi:10.1038/sj.onc.1210607
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Reference Table: List of proteins that have been identified in hMOF-containing complexes (6).
Name
Full name
Method for
interaction
Domains
References
hMSL1*
Male-specific lethal-1
Coiled coil, PEHE domain
Affinity purification,
Co-IP
(Smith et al., 2005;
Mendjan et al., 2006)
hMSL2*
Male-specific lethal-2
RING finger, PHD finger
Affinity purification,
Co-IP
(Smith et al., 2005;
Mendjan et al., 2006)
hMSL3a/MSL3L1*
hMSL3c/MSL3L1c
Male-specific lethal-3
Chromobarrel, MRG homology
shorter version lacking the
chromobarrel
Affinity purification,
Co-IP, GST-pull down
(Smith et al., 2005;
Taipale et al., 2005;
Mendjan et al., 2006)
MRG15
MORF-related gene on
chromosome 15
Chromodomain, MRG domain
Co-IP
(Pardo et al., 2002)
(Smith et al., 2005;
Mendjan et al., 2006)
NSL1/hMSL1v1*(KIAA1267/LOC284058)
Non-specific lethal-1
Coiled coil, PEHE domain
Affinity purification,
Co-IP
NSL2* (FLJ20436)
Non-specific lethal-2
Two domains rich in cysteine and
histidine
Affinity purification,
Co-IP
(Mendjan et al., 2006)
NSL3* (FLJ10081)
Non-specific lethal-3
Affinity purification,
Co-IP
(Mendjan et al., 2006)
Affinit purification,
Co-IP
(Dou et al., 2005;
Mendjan et al., 2006)
/
-hydrolase fold
HCF-1
Host cell factor 1
Six kelch repeats –
domains, Fn3
OGT
O-linked
-Nacetylglucosaminetransferase
TPR, glycosyltransferase
Affinity purification
(Mendjan et al., 2006)
WDR5 or BIG-3*
WD repeat domain 5
Seven WD40 repeats
Affinity purification
(Dou et al., 2005;
Mendjan et al., 2006)
TPR*
Translocated promoter
region
Coiled coil
Affinity purification
(Mendjan et al., 2006)
MCRS2/p78*
Microspherule protein 2
Forkhead associated domain (FHA)
Affinity purification
(Dou et al., 2005;
Mendjan et al., 2006)
PHF20*
PHD finger protein 20
2 Tudor domain, PHD finger,
C2H2 – type zinc finger
Affinity purification
(Dou et al., 2005;
Mendjan et al., 2006)
PHF20L1*
PHD finger protein 20 like
1
2
Tandem Tudor domain
Affinity purification
(Mendjan et al., 2006)
ILF1/FOXK2
Interleukin enhancer
binding factor 1
Fork head DNA binding domain
Affinity purification
(Mendjan et al., 2006)
MLL1
Mixed-lineage leukemia
PHD finger and bromodomain
Affinity purification,
Co-IP, GST-pull down
(Dou et al., 2005)
ATM
Ataxia telangiectasia
mutated
FAT, FATC, PI3Kc
Two-hybrid, Co-IP,
GST-pull down
(Gupta et al., 2005)
p53
TAD, DBD, OD and a proline rich
domain
GST-pull down
(Dou et al., 2005; Sykes et
al., 2006)
p53
8
propeller