Understanding the roles of histone demethylation in germline maintenance

Harvard Medical School, Boston MA

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Abstract

DESCRIPTION (provided by applicant): Histone tails are the targets of an enormous variety of covalent but reversible modifications that affect chromatin structure and direct the activities of specific protein complexes to sites along the chromosome for specialized functions. The methylation of histone lysines has a function in transcription and DNA damage response, where defects can result in cancers, and also plays a particularly prominent role in crossover formation during meiosis, where defects can result in a variety of birth abnormalities, infertility and miscarriages. The deregulation or mutation of lysine demethylases has been associated with a variety of cancers, highlighting their importance in maintenance of genomic integrity. Three putative LSD1/2 (human lysine specific demethylase) homologs exist in C. elegans (SPR-5, AMX-1, and T08D10.2), and our preliminary studies indicate that these proteins are important for proper maintenance of the C. elegans germline and may play a direct role for in DNA damage repair. The goal of this proposal is to develop a mechanistic understanding of the way histone methylation orchestrates critical chromosomal events during meiosis. The experiments we propose will provide insight into the important yet poorly-defined roles that demethylation plays in promoting meiotic function and maintenance of genomic integrity in the germline. The C. elegans germline is an ideal system for studying meiosis and germline maintenance due to the unique spatial and temporal organization of meiotic nuclei throughout the gonad and the range of tools that have been developed to track important meiotic events in the germline. Furthermore, given the high degree of homology shared throughout genes and biological pathways between C. elegans and mammals this work will be very informative in understanding germline maintenance in higher eukaryotes as well. We will use combined genetic, biochemical, and cytological approaches to functionally characterize the LSD1/2-like proteins in C. elegans and determine both the specific substrates for their histone demethylase activity and their gene targets. Specifically, we propose: 1) To biochemically and biologically determine the substrate specificity of the C. elegans LSD1/2-like proteins both in vitro and in vivo, and determine their genomic binding profiles throughout meiosis; 2) To gain mechanistic insight into the role of the LSD1-like proteins in DNA damage repair by assessing what is required for them to relocalize in response to induced DNA damage and determining whether they are required for the DNA repair machinery to assemble at sites of damage; and 3) To carry out a targeted RNAi screen to identify proteins that functionally interact with and/or regulate the activity of the LSD1/2-like proteins, giving us insight into the network of proteins involved in regulating histone methylation status during meiosis. This work will provide mechanistic insights into the roles of histone demethylases both in germline maintenance and meiosis that are likely to be conserved in humans.