Using the formation of a virus particle (virion) from SV40 minichromosomes as a model for the generation of repressive chromatin by nucleosome remodeling, we will characterize how the combination of nucleosome location and associated histone modifications controls gene expression. Our central hypothesis is that the nucleosome location and the histone modifications associated with the nucleosome directly contribute to reorganization.
The specific aims are (1) To characterize the changes in nucleosome positioning and corresponding histone modifications during the encapsidation of minichromosomes late in infection and (2) To determine which transcriptional regulatory sequences direct nucleosome positioning and histone modifications during encapsidation. For comparison purposes bulk nucleosome locations will be determined from micrococal nuclease digestion of chromatin from minichromosomes and disrupted virions. The location of nucleosomes containing specific histone modifications along with all of the other nucleosome positions in each sample of chromatin will be obtained using our modified ChIP-Seq procedure, which we developed. Intact SV40 chromatin will be immune selected with antibody to a particular histone modification bound to agarose, the ChIP chromatin purified on the agarose, and fragmented by micrococcal nuclease digestion. The DNA present in the agarose-bound chromatin fragments and the free fragments will be separately purified, libraries prepared, and paired-end sequenced using an Illumina MiSeq. The location of nucleosomes will then be determined from a bioinformatics analysis of the sequencing reads in the parallel libraries. SV40 chromatin will be obtained from wild-type and mutant minichromosomes and encapsidation intermediates and recombinant constructs containing specific regulatory elements within a defined chromatin reporter region. The changes in nucleosome location and histone modifications will then be correlated with the process of encapsidation and the presence of specific DNA elements. The proposed studies are innovative because for the first time we will be characterizing both nucleosome location and histone modifications in the same nucleosomes while remodeling to repress transcription is occurring as part of virion formation. The work is also significant since linking histone modifications to nucleosome location would advance our understanding of how nucleosome location is regulated. Because SV40 is also a model for closely related human pathogens understanding epigenetic repression by nucleosome positioning may lead to novel therapeutic approaches to address the disease caused by the pathogens.
The proposed studies will be the first to investigate the impact of integral histone modifications on the reorganization of nucleosomes, which results in the repression of transcription using the formation of the SV40 virus particle as a model. The results will add to our understanding of chromatin remodeling, in the context of repression of transcription through the control of accessibility of chromatin for critical transcriptional factors. Infection by SV40 has served as an excellent model system to study the regulatory events in eukaryotic molecular biology including replication and transcription. The studies outlined in this proposal will enhance our understanding of the role of histone modifications and nucleosome location acting coordinately in controlling accessibility of DNA for binding by regulatory factors. SV40 has also served as a useful model for other related viruses that are more difficult to work with experimentally. As such the results will help to identify areas of productive research on these other viruses which may yield therapeutic benefits.
| Balakrishnan, Lata; Milavetz, Barry (2017) Epigenetic Analysis of SV40 Minichromosomes. Curr Protoc Microbiol 46:14F.3.1-14F.3.26 |
| Kumar, Meera Ajeet; Christensen, Kendra; Woods, Benjamin et al. (2017) Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection. Virology 503:62-69 |
