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所必需的，但对生育力至关重要，因此对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是如何获得和保持这些永生和全能的特性的。我们的研究结合了联合收割机强大的遗传学，基因组学和分子生物学方法的模式系统秀丽隐杆线虫。该建议集中在生殖细胞中染色质状态和基因表达模式的调节。我们以前确定了四个C。线虫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作为一个复合物起作用，将抑制性组蛋白修饰、组蛋白H3在Lys 27上的甲基化集中在X上。相比之下，MES-4显著集中在常染色体上，在那里它甲基化Lys 36上的组蛋白H3。尽管MES-4在X染色体上明显缺失，但去除MES-4主要导致X染色体上基因的去沉默。我们对X沉默的工作模型是MES-2/3/6作用直接抑制基因表达，而MES-4通过排斥来自常染色体区域的阻遏物并将其作用集中在X上而在远处起作用。该提案的目的是：1）通过调查候选基因并使用强大的无偏见遗传筛选来测试该模型并识别MES调节中的新参与者。2)在基因水平上了解MES-4及其H3 Lys 36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复合物如何使MES-4远离X染色体。这些研究将使用染色质免疫沉淀，然后使用平铺阵列（ChIP芯片）方法。3)获得MES-2/3/6及其H3 Lys 27甲基标记在基因组中分布的高分辨率视图，并测试常染色体MES-4是否参与将MES-2/3/6集中在X上。这一目标也将利用ChIP芯片技术。除了对C.通过对线虫生殖细胞的研究，我们的研究将揭示H3 Lys 27和H3 Lys 36甲基化在不同物种中的不同作用和调节，细胞如何调节大结构域和整个染色体的染色质状态，例如在剂量补偿期间，以及MES蛋白的哺乳动物同源物如何有助于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.