Effect of Adenovirus E1A Oncogene on DNA Replication Dynamics
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|ClinicalTrials.gov Identifier: NCT03325517|
Recruitment Status : Unknown
Verified October 2017 by Heba Momen kamel, Assiut University.
Recruitment status was: Not yet recruiting
First Posted : October 30, 2017
Last Update Posted : October 30, 2017
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.
- Using standard assays the investigators will determine whether E1A binds to HBO1 to induce its HAT activity
- 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.
|Condition or disease|
|Study Type :||Observational|
|Estimated Enrollment :||50 participants|
|Official Title:||Effect of Adenovirus E1A Oncogene on DNA Replication Dynamics|
|Estimated Study Start Date :||November 1, 2017|
|Estimated Primary Completion Date :||September 1, 2018|
|Estimated Study Completion Date :||September 1, 2019|
- DNA re-replication indicated by the number of cells containing >4N DNA content using flow cytometer [ Time Frame: 1 year ]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.
Biospecimen Retention: Samples With DNA