Investigation of roles and mechanisms of DNA and histone modification networks in trans-generational epigenetic inheritance

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Summary: Most heritable information is transmitted by DNA, following Mendelian inheritance patterns; but some traits, such as longevity, fertility, disease susceptibility, and obesity, can be inherited non-genetically in several model organisms. The underlying molecular mechanisms of trans-generational epigenetic transmission remain unclear, but chromatin changes may play a role. Chromatin is composed of DNA and histone proteins, both of which are extensively chemically modified. These modifications are recognized by so called “reader” proteins, and they regulate chromatin structure and function, together with the enzymes that add and remove modification marks. Previous work identified a fascinating trans-generational epigenetic inheritance phenomenon in C. elegans, whereby worms lacking spr-5, the human homolog of the histone H3K4me2 demethylase LSD1, do not exhibit sterility initially; however, successive generations lacking spr-5 display increasing infertility concomitant with global accumulation of H3K4me2. This progressive phenotype can be reversed by introducing a single copy of SPR-5, but how this epigenetic memory is transmitted across generations is still unknown. In many species DNA methylation at the 5th position of cytosine (5mC) is well- documented as one of the major regulatory mechanisms of epigenetic inheritance. However, 5mC is absent in C. elegans, as is the enzymatic machinery that installs methylation on cytosine, raising the question as to how epigenetic inheritance is accomplished in species such as C. elegans that lack cytosine methylation. Our recent work has uncovered an exciting new facet to epigenetic inheritance in C. elegans: we have discovered a novel DNA modification-- methylation of adenine at the 6th position (6mA)-- and have also identified the enzymes responsible for adding and removing this mark. Interestingly, this modification accumulates over the generations in mutants lacking SPR-5, paralleling the increase in H3K4me2 and the decrease in fertility, suggesting that 6mA participates in a network of epigenetic interactions that regulate germline immortality. However, little is known about how 6mA is regulated, its mechanism of accumulation in spr-5 mutants, or its role in regulating gene expression or chromatin architecture in a manner that affects fertility in spr-5 mutants. In this application, we will investigate the genome-wide distribution of 6mA (paying special attention to its accumulation in late-generation spr-5 worms) and its effects on transcription in wild-type and spr-5 mutants; determine the sites of action of the 6mA methyltransferase and demethylase as well as their means of regulation, particularly in the context of maintaining fertility; and finally identify proteins that recognize 6mA to promote downstream events such as appropriate germline gene expression programs. The proposed studies are built upon the recent discovery of 6mA and are expected to elucidate an unappreciated mode of gene regulation, providing insight into mechanisms that control epigenetic inheritance and informing studies in other species.