Adenovirus Clinical Trial
Official title:
Effect of Adenovirus E1A Oncogene on DNA Replication Dynamics
BACKGROUND:
The 243 amino acid E1A encoded by the left end of the human adenovirus (Ad) type 2 or 5
genome has been studied in various contexts including as a model cooperating oncoprotein , an
apoptosis inducing protein, and as a therapeutic oncolytic protein. All of these properties
are associated with its capacity to rapidly induce S phase in a variety of cells. E1A
orchestrates most of these effects by interacting with an array of chromatin remodeling
complexes, including the Rb family proteins, and the HAT proteins p300 and CBP.
The Myst family protein HBO1 (Myst2, KAT7) is a histone acetyl transferase that plays a major
role in replication initiation and also contributes to DNA re-replication. HBO1 directly
interacts with Cdt1 and functions as a coactivator of Cdt1 in replication initiation. It also
associates with replication origin and stimulates origin activation by acetylating H4 K5, K8,
and K12. Overexpression of HBO1 induces DNA re-replication.
SPECIFIC AIM OF THE STUDY: The Specific Aim of this study is to determine whether or not the
stimulation of HBO1 activity by E1A plays a role in deregulated DNA replication, and if it
does, to determine the mechanism.
1. Using standard assays the investigators will determine whether E1A binds to HBO1 to
induce its HAT activity
2. The investigators will determine whether E1A stimulation of HAT activity of HBO1
contributes to DNA re-replication.
RESEARCH DESIGN AND METHODS:
First, the investigators will determine whether E1A associates with replication origins and
whether this association requires HBO1. The investigators will use the MCM4 origin which maps
in the intergenic region between PRKDC and (Protein Kinase, DNA-Activated, Catalytic
Polypeptide) and MCM4 genes.
The investigators will transfect U2OS cells with plasmids expressing relevant proteins then
determine their occupancy in origin sequences using ChIP assays. Plasmids expressing epitope
tagged WT HBO1 or mutant derivatives along with plasmids expressing WT or mutant E1A proteins
will be expressed in U2OS cells, then occupancy of these proteins on origin regions will be
quantified using antibodies as appropriate. Typically in these experiments, using relevant
antibodies, ChIP assays are performed with primer pairs encompassing origin regions and also
regions that are far from origin. Occupancy of initiation factors are increased several fold
in the origin region as compared to that of 2KB upstream or downstream regions. Loading of
MCM complex along with Cdt1 onto the origins is an indication that initiation of replication
occurs in that origin and usually assayed in ChIP-re-ChIP assays as follows: First, loading
of HBO1 to the origins will be confirmed using epitope specific antibodies in the first ChIP.
The anti-HBO1 precipitates will be re-ChIPed with anti-MCM3 antibodies. Loading of MCM3
helicase to origins occurs after Cdt1 binding and depends on HBO1 HAT activity. Normal
amounts of MCM3 will be detected after re-ChIP-ing in E1A+ control samples. If reduced amount
of MCM3 is recovered in reChIP assays when mutant E1A or HBO1 mutant (e.g. HBO1 G435A) is
used, it would indicate that stimulation of HAT activity by E1A is critical for maximal
origin activity in E1A+ cells. This type of assay has considerable flexibility in that mutant
proteins can be rapidly assayed. This ChIP-reChiP assays will repeated in different
combinations to determine the E1A loading.
These results will be extended to virus infection assays. G1 specific cells isolated by drug
treatment will be infected with Ad vectors expressing epitope tagged proteins as appropriate.
Association of E1A and HBO1 and their mutant derivatives will be determined. This assay will
allow us to confirm the effect of E1A stimulated HAT activity in origin firing and study the
effects of E1A on origin firing, if any, other than increasing the HAT activity of HBO1.
BACKGROUND:
TThe 243 amino acid E1A protein (also known as small E1A protein, transforming E1A protein)
encoded by the left end of the human adenovirus (Ad) type 2 or 5 genome has been studied in
various contexts including as a model cooperating oncoprotein, an apoptosis inducing protein,
and as a therapeutic oncolytic protein. All of these properties are associated with its
capacity to rapidly induce S phase in a variety of cells. E1A orchestrates most of these
effects by interacting with an array of chromatin remodeling complexes, including the Rb
family proteins, and the HAT proteins p300 and CBP.
The induction of E2F by E1A-Rb-HDAC interactions is well documented whereas the consequences
of E1A-p300/CBP interactions in cell cycle progression are not clear. Previous studies
demonstrated that p300/CBP prevents premature entry of quiescent cells into S phase by
repressing c-Myc transcription through a tripartite repressor complex consisting of p300,
YY1, HDAC3. E1A binds to p300 and dissociates the repressor complex to induce S phase.
Induction of c-Myc by this mechanism contributes to the induction of DNA damage response and
aberrant cellular DNA replication which leads to genomic instability. In a recently published
study, it was shown that E1A induces the pivotal DNA replication initiation factor Cdt1 to
high levels resulting in increased replication origin activity and in late S phase, induction
of DNA damage response and cellular DNA re-replication. Using the single molecule DNA fiber
assay, it was discovered that E1A induces significant changes in the dynamics of DNA
replication and appears to induce global changes in origin activation.
Cdt1 is a pivotal DNA replication initiation factor whose levels oscillate during cell cycle
progression. In late G1, its levels rise to initiate DNA replication. At the beginning of S
phase it is promptly degraded by the E3 ligase so that origins that are already fired once do
not fire again in S phase and re-replication within a single cell cycle does not occur.
Impaired degradation of Cdt1 in S phase by the E3 ligase is the major mechanism by which the
cellular DNA undergoes re-replication. It was discovered that in E1A expressing cells Cdt1
levels remain high in S phase suggesting an inefficient degradation of Cdt1 which may
contribute to extensive re-replication of cellular DNA that the investigators observed in
late S phase.
The Myst family protein HBO1 (Myst2, KAT7) is a histone acetyl transferase that plays a major
role in replication initiation and also contributes to DNA re-replication. HBO1 directly
interacts with Cdt1 and functions as a coactivator of Cdt1 in replication initiation. It also
associates with replication origin and stimulates origin activation by acetylating H4 K5, K8,
and K12. Overexpression of HBO1 induces DNA re-replication. The enrichment of chromatin
modifications including acetylation of H4 histones at the origins strongly influences origin
activation. Thus, E1A in the absence of a multitude of serum stimulated proliferation
signals, forces cells to enter S phase using several compensatory mechanisms. Uncontrolled
cellular DNA replication program causes polyploidy that is the hallmark of cancer. It is
exciting that a viral oncogene such as E1A alters the cellular DNA replication program. This
raises a number of important questions related to the mechanism of replication stress induced
by a viral oncogene. For example, how does E1A induce DNA re-replication? Does E1A
stimulation of HBO1 alter initiation mechanism and whether HBO1 mediated chromatin
modifications at the origins promote re-replication?
SPECIFIC AIM OF THE STUDY:
Our Specific Aim is to determine whether or not the stimulation of HBO1 activity by E1A plays
a role in deregulated DNA replication, and if it does, to determine the mechanism.
1. Using standard assays the investigators will determine whether E1A binds to HBO1 to
induce its HAT activity
2. Using transient and virus infection assays, the investigators will determine whether
stimulation of HBO1 HAT activity by E1A enhances the pre-replicative complex (Pre-RC)
formation and whether E1A induced HAT activity qualitatively and quantitatively changes
the H4 acetylation at the origin regions.
3. the investigators will determine whether E1A stimulation of HAT activity of HBO1
contributes to DNA re-replication.
RESEARCH DESIGN AND METHODS:
The goal of this study is to determine whether stimulation of HBO1 HAT activity by E1A (i)
enhances initiation (ii) induces qualitative and quantitative changes in acetylation of H4
K5, K8 and K12 at the origin region, and (iii) enhances re-replication that occurs in E1A+
cells. If increased HAT activity can stimulate one or more of these functions, it could
explain how this property of E1A would contribute to the induction of aberrant DNA
replication.
1. E1A binding to WT and HAT deficient HBO1.
the investigators will first determine whether E1A directly binds to HBO1 by performing
the GST binding and in vivo co-IP experiments. If binding is confirmed, the
investigators will map the binding sites on both proteins. the investigators will then
isolate mutants of both proteins that abolish the interactions. All E1A mutants will be
evaluated for their interactions with Rb and p300 and only mutants that retain their
capacity to bind to these proteins will be used. It will be necessary to isolate an HBO1
mutant that retains E1A binding capacity but lacks the HAT activity. A widely used HAT
deficient mutant G435A will be used in these studies. If E1A binds to this mutant, it
will be a valuable mutant to confirm that E1A induced HAT activity is critical for
stimulation of replication initiation. If this HBO1 mutant does not bind to E1A, it will
be important to isolate new HBO1 mutants which retain E1A binding capacity but lack HAT
activity.
2. Role of E1A induced HBO1 HAT activity in origin activation:
First, the investigators will determine whether E1A associates with replication origins
and whether this association requires HBO1. the investigators will use the MCM4 origin
which maps in the intergenic region between PRKDC and (Protein Kinase, DNA-Activated,
Catalytic Polypeptide) and MCM4 genes. First, the investigators will transfect U2OS
cells with plasmids expressing relevant proteins then determine their occupancy in
origin sequences using ChIP assays. Plasmids expressing epitope tagged WT HBO1 or mutant
derivatives along with plasmids expressing WT or mutant E1A proteins will be expressed
in U2OS cells, then occupancy of these proteins on origin regions will be quantified
using antibodies as appropriate. Typically in these experiments, using relevant
antibodies, ChIP assays are performed with primer pairs encompassing origin regions and
also regions that are far from origin. Occupancy of initiation factors are increased
several fold in the origin region as compared to that of 2KB upstream or downstream
regions. Loading of MCM complex along with Cdt1 onto the origins is an indication that
initiation of replication occurs in that origin and usually assayed in ChIP-re-ChIP
assays as follows: First, loading of HBO1 to the origins will be confirmed using epitope
specific antibodies in the first ChIP. The anti-HBO1 precipitates will be re-ChIPed with
anti-MCM3 antibodies. Loading of MCM3 helicase to origins occurs after Cdt1 binding and
depends on HBO1 HAT activity. Normal amounts of MCM3 will be detected after re-ChIP-ing
in E1A+ control samples. If reduced amount of MCM3 is recovered in reChIP assays when
mutant E1A or HBO1 mutant (e.g. HBO1 G435A) is used, it would indicate that stimulation
of HAT activity by E1A is critical for maximal origin activity in E1A+ cells. This type
of assay which has been successfully used by others has considerable flexibility in that
mutant proteins can be rapidly assayed. This ChIP-reChiP assays will repeated in
different combinations to determine the E1A loading.
These results will be extended to virus infection assays. G1 specific cells isolated by
drug treatment will be infected with Ad vectors expressing epitope tagged proteins as
appropriate. Association of E1A and HBO1 and their mutant derivatives will be
determined. This assay will allow us to confirm the effect of E1A stimulated HAT
activity in origin firing and study the effects of E1A on origin firing, if any, other
than increasing the HAT activity of HBO1. For example, c-Myc induction by E1A does not
involve HAT activity.
3. Effects of E1A induced HBO1 HAT activity in the acetylation of H4, K5, K8 and K12.
To assess this, the investigators will ChIP the chromatin prepared from virus infected
cells with the origin specific primers and epitope specific antibodies as appropriate to
detect origin loading of HBO1. the investigators will then re-Chip the ChIP precipitates
using anti-H4 tetra-Ac antibodies to detect K5, K8, and K12 acetylation. In cells
expressing E1A and HBO1, H4 acetylation will be increased considerably as compared to
cells infected with HBO1 virus alone. H4 acetylation may be affected depending on the
mutants used.
4. E1A stimulated HBO1 HAT activity in re-replication.
Overexpression of HBO1 has been shown to induce DNA-replication. To determine whether
E1A uses HBO1 in inducing re-replication by stimulating the HBO1 HAT activity, the
investigators will infect S phase enriched cells (isolated by double thymidine block)
with Ad viruses expressing the WT or mutant E1A proteins along with Ad vectors
expressing HBO1 variants (e.g. HAT inactive), allow the infection to proceed as needed
then quantify the population of cells containing >4N DNA content using flow cytometer.
If E1A simulated HBO1 HAT activity is important for re-replication HAT defective HBO1
mutants will not induce re-replication even in the presence of E1A. E1A mutants that
cannot bind to HBO1 will also show a similar phenotype.
5. E1A effects on H4 acetylations in re-replicating origins:
In order to study the H4 acetylations in re-replicating origins, the investigators need
to know which of cellular origins undergo re-replication in E1A+ cells in late S phase.
the investigators are planning to identify the origins activated in E1A+ cells including
those that re-replicate. Once the investigators identify one or two re-replicating
origins, the investigators will quantify the acetylations using these origins in cells
expressing epitope tagged WT and mutant HBO1 alleles and E1A. the investigators
anticipate qualitative or quantitative (or both) in H4 acetylations due to E1A.
6. Expected results and pitfalls:
At the end of all the proposed experiments, the investigators expect to determine whether
stimulation of HBO1 HAT activity by E1A is critical for origin activation in E1A+ cells and
if E1A induced HAT activity has a role in re-replication. The investigators do not anticipate
any technical or other problems including obtaining relevant reagents.
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