Dissecting the roles of histone H3 lysine 27 mono-methylation in DNA replication

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Genomic DNA is compacted by association with histone proteins. Covalent modifications of histones affect DNA compaction and therefore accessibility to other proteins that need to contact the DNA. One example is proteins involved in replication of the genomic DNA. The overall impact of this project is to understand how certain covalent modifications contribute to the regulation of DNA replication. In addition, delineating patterns of histone modifications will be a resource for other investigators that study basic mechanisms of DNA organization. Data generated by this project will be actively distributed to the wider research community through the Tetrahymena Genome Database (TGD: http://ciliate.org/) and the Tetrahymena Gene Expression Database (TGED: http://tged.ihb.ac.cn/). This will be part of an effort to promote the unicellular eukaryote Tetrahymena as a model organism for scientific research, as well as a teaching tool for undergraduate students. Undergraduate students will also contribute to the research program, mainly through collaboration with the Undergraduate Research Opportunity Program (UROP) at the University of Michigan. A collaboration within the framework of the Ciliate Genome Consortium (CGC: http://tet.jsd.claremont.edu), an NSF-funded collaborative research initiative involving undergraduate students from diverse institutes, will also be pursued. This project will involve undergraduate students in many aspects of biology research at a level often reserved for graduate students, providing them experiences that will help them succeed in the pursuit of professional and graduate school training.The histone modification that is at the center of this project is histone H3 lysine 27 methylation, a modification that is conserved in protozoa, metazoa and plants. This modification has long been associated with transcriptional repression and heterochromatin formation. Unexpectedly, a role in DNA replication has been recently revealed for histone H3 lysine 27 mono-methylation (H3K27me1). Deleting TXR1, the enzyme responsible for this methylation event in Tetrahymena, leads to DNA replication stress as well as H3K27me1 deficiency. Phenotypic analysis of ΔTXR1 cells provides an entry point to dissect the molecular connection between histone modifications and DNA replication. By identifying histone modifications that correlate with DNA synthesis both temporally and spatially, this project will shed light on the chromatin environment that supports DNA replication. TXR1 mutagenesis will dissect its function, revealing mechanisms for its targeting to active replication sites, while direct mutation of H3 K27 will help to elucidate the connection between H3K27me1 and DNA replication. Successful completion of this project will secure insights into the molecular mechanisms by which histone modifications control regulation of DNA replication.