The establishment and maintenance of heterochromatin and DNA methylation is essential for the proper growth and development of all organisms. Specifically, heterochromatin and DNA methylation are essential for X- chromosome inactivation, chromosome segregation, and parasitic gene silencing in humans. However, the mechanisms controlling formation of heterochromatin, and subsequent DNA methylation, are presently unclear. In order to fully understand these critical human developmental processes, the mechanisms underlying the formation of heterochromatin and DNA methylation must be discerned. Heterochromatic regions of the genome are distinguished from their actively transcribed euchromatic counterparts by several covalent modifications. Namely, heterochromatic DNA and associated histone proteins are methylated. While several proteins have been identified that catalyze the methylation of DNA and histones, the regulation of these methyltransferase proteins is only now being appreciated. Studies in the model organism Neurospora crassa, a filamentous fungus, have been critical to understanding the formation of heterochromatin. Neurospora shares conserved DNA methylation machinery with humans, but unlike many higher eukaryotes, this methylation machinery is simple yet dispensable. Recent work has identified a protein that is known to be critical for nuclear transport as being important to establish both histone and DNA methylation, indicating that this protein has a critical role in regulating the methylation machinery. This proposal will focus on characterizing the role of the nuclear transport protein in DNA methylation. DamID experiments using translational fusions to the bacterial DNA Adenine Methylase (dam) gene will analyze the genomic localization of this protein. The influence of this nuclear transport protein on the global activity of the methylation machinery will be analyzed by western blotting experiments with tagged components of the DNA methylation machinery. Translational fusions of this protein to Green Fluorescent Protein (GFP) will analyze the cellular localization of this protein. Co-immunoprecipitation experiments will analyze the ability of this nuclear transport protein to interact with the H3K9me3 machinery. Moreover, the ability of the methylation machinery to influence the genomic localization of the nuclear transport protein will be investigated. Lastly, known interactors of the nuclear transport protein will be examined for their role in DNA methylation. By characterizing the genes required for the regulation of the DNA methylation machinery, we will be able to understand how heterochromatin is properly established in humans. In addition, identifying putative genes that could become mutated for the progression of cancer is essential to develop treatments or prevention strategies for cancer patients.
Many human developmental processes require a chemical modification of DNA, termed methylation, for their proper function. Experimentation in the model fungus Neurospora crassa, which utilizes a dispensable DNA methylation system that is similar to human methylation, can help us understand how individual genes control DNA methylation. This proposal will characterize the role of a essential Neurospora gene that regulates the fungal DNA methylation machinery, allowing us to comprehend the DNA methylation regulation that is critical for human development.
|Galazka, Jonathan M; Klocko, Andrew D; Uesaka, Miki et al. (2016) Neurospora chromosomes are organized by blocks of importin alpha-dependent heterochromatin that are largely independent of H3K9me3. Genome Res 26:1069-80|
|Klocko, Andrew D; Rountree, Michael R; Grisafi, Paula L et al. (2015) Neurospora importin Î± is required for normal heterochromatic formation and DNA methylation. PLoS Genet 11:e1005083|