Control of early germline development in C. elegans

描述（由申请人提供）：生殖细胞在动物发育中起着独特的作用-它们不是生物体生存能力所必需的，但对于生育能力和物种的延续至关重要。需要特殊的控制机制来确保生殖细胞从一代存活到下一代，并能够产生全新的生物体。我们试图了解生殖细胞如何获得和保持这些不朽和全能性的特性。我们的研究在秀丽隐杆线虫模型系统中结合了强大的遗传学，基因组学和分子方法。本研究主要关注生殖细胞中染色质状态和基因表达模式的调控。我们之前发现了四种秀丽隐杆线虫的MES蛋白是生殖细胞存活和生育所必需的，并表明它们的主要作用是沉默X染色体。MES-2、MES-3和MES-6作为一个复合体，集中抑制组蛋白修饰，即组蛋白H3在Lys27和x上的甲基化。相反，MES-4显著地集中在常染色体上，在那里它甲基化Lys36上的组蛋白H3。尽管它明显不在X染色体上，但去除MES-4主要导致X染色体上基因的沉默。我们的X沉默工作模型是，MES-2/3/6作用直接抑制基因表达，而MES-4作用在远处，通过排斥来自常染色体区域的抑制因子并将其作用集中在X染色体上。本提案的目的是：1)通过调查候选基因和使用强大的无偏遗传筛选来测试该模型并确定MES调节的新参与者。2)在基因水平上了解MES-4及其H3Lys36甲基标记的位置，并研究MES-4如何被招募到这些位点以及MES-2/3/6复合体如何使MES-4远离X染色体。这些研究将使用染色质免疫沉淀和贴片阵列（ChIP-chip）方法。3)获得MES-2/3/6及其H3Lys27甲基标记在基因组中的高分辨率分布，并检测常染色体MES-4是否参与了MES-2/3/6在x染色体上的集中。这一目标也将利用ChIP-chip技术。除了了解秀丽隐杆线虫生殖细胞的发育策略外，我们的研究还将揭示H3Lys27和H3Lys36甲基化在物种间的不同作用和调控，细胞如何调节大结构域和整个染色体的染色质状态，例如在剂量补偿过程中，以及MES蛋白的哺乳动物同源物如何促进干细胞生物学和癌症。

DESCRIPTION (provided by applicant): 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.