Using the Simian Virus 40 (SV40) chromosome as a model for eukaryotic chromatin, the proposed research will address the role of histone hyperacetylation at an active replication fork and during post-replication chromatin maturation within the regulatory region of a chromosome whose biological function changes over the course of infection. The studies will answer fundamental questions concerning the mechanism of eukaryotic replication and the relationship between eukaryotic replication and gene regulation which will be potentially applicable to the therapy of a wide variety of diseases including viral infections and cancer.
The specific aims of the application are 1. Characterization of histone hyperacetylation in the vicinity of a replication fork and 2. Characterization of histone hyperacetylation associated with post-replicative chromatin remodeling within the SV40 promoter/regulatory region. Histone hyperacetylation in the vicinity of a replication fork will be characterized in replicating SV40 chromosomes either immune selected with antibodies which recognize proteins found only at the fork or labeled with biotin conjugated deoxyribonucleotides. In both strategies the status of histone hyperacetylation at various sites on the pre- and post-replicative sides of the replication fork will be determined using a combination of biophysical separation, inhibition of replication by chemicals and siRNAs, and chromatin fragmentation in combination with chromatin immunoprecipitation analyses including two which we have developed: Immune Selection and Fragmentation (ISF) and Immune Selection Fragmentation and Immunoprecipitation (ISFIP). Post-replication chromatin maturation within the regulatory region will be characterized in SV40 chromosomes and confirmed in replication-competent plasmids carrying portions of the SV40 regulatory region. Maturation occurring during replication will be determined in replication intermediates, while maturation occurring after replication will be determined by comparing histone hyperacetylation present in terminating replicating chromosomes to hyperacetylation present in chromosomes competent for late transcription and encapsidation. Immune selection with antibodies specific for replicating, transcribing, and encapsidating chromosomes will be used in conjunction with various ChIP techniques to determine the status of histone hyperacetylation in the chromosomes and plasmids.
The proposed studies will be the first detailed characterization of histone hyperacetylation during the eukaryotic replication process and will directly link changes in histone hyperacetylation to specific events occurring during the replication process. The results obtained will significantly add to our knowledge of the role of replication in eukaryotic gene regulation and be potentially useful in the development of therapeutics for viral infections and cancers. ? ? ?
| Milavetz, Barry; Kallestad, Les; Gefroh, Amanda et al. (2012) Virion-mediated transfer of SV40 epigenetic information. Epigenetics 7:528-34 |
| Balakrishnan, Lata; Gefroh, Amanda; Milavetz, Barry (2010) Histone H4 lysine 20 mono- and tri-methylation define distinct biological processes in SV40 minichromosomes. Cell Cycle 9:1320-32 |
| Balakrishnan, Lata; Milavetz, Barry (2010) Decoding the histone H4 lysine 20 methylation mark. Crit Rev Biochem Mol Biol 45:440-52 |
