Germ cells serve a unique role in animal development - they are not required for organism viability but are essential for fertility and thus for perpetuation of species. Special control mechanisms are required to ensure that germ cells survive from one generation to the next and are able to produce entire new organisms. We seek to understand how germ cells acquire and preserve these properties of immortality and totipotency. Our studies combine powerful genetic, genomic, and molecular approaches in the model system Caenorhabditis elegans. This proposal focuses on regulation of chromatin states and gene expression patterns in germ cells. We previously identified four C. elegans MES proteins as being required for germ cell survival and fertility and showed that their major role is silencing the X chromosomes. MES-2, MES-3, and MES-6 function as a complex to concentrate a repressive histone modification, methylation of histone H3 on Lys27, on the Xs. In contrast, MES-4 is dramatically concentrated on the autosomes, where it methylates histone H3 on Lys36. Despite its apparent absence from the X, removal of MES-4 leads primarily to desilencing of genes on the X. Our working model for X silencing is that MES-2/3/6 action directly represses gene expression and that MES-4 acts at a distance, by repelling repressors from autosomal regions and focusing their action on the Xs.
The aims of this proposal are to: 1) Test that model and identify new participants in MES regulation by investigating candidate genes and using a powerful unbiased genetic screen. 2) Learn at the gene level where MES-4 and its H3Lys36 methyl marks are located and investigate how MES-4 is recruited to those sites and how the MES-2/3/6 complex keeps MES-4 off the X chromosomes. These studies will use chromatin immunoprecipitation followed by tiling array (ChIP-chip) approaches. 3) Gain a high resolution view of the distribution of MES-2/3/6 and its H3Lys27 methyl marks across the genome and test whether autosomal MES-4 participates in concentrating MES-2/3/6 on the Xs.
This aim will also take advantage of ChIP-chip technology. In addition to insights into developmental strategies used in C. elegans germ cells, our studies will shed light on the diverse roles and regulation of H3Lys27 and H3Lys36 methylation across species, how cells regulate the chromatin state of large domains and entire chromosomes, for instance during dosage compensation, and how mammalian homologs of the MES proteins contribute to stem cell biology and cancer.
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