Analysis of Nematode Sex Determination and Dosage Compensation

项目总结 有人建议进行研究，以剖析最基本的二元发展决策之一 后生动物制造：他们的性行为。线虫线虫通过计数X-X来非常精确地确定性别- 相对于常染色体组的染色体数目(X：A信号)：1X：2A(0.5)和2X：3A(0.67)的比率 标志着雄性的命运，而3X：4A(0.75)和2X：2A(1.0)的比例标志着两性的命运。我们发现 更多关于X的性质和作用：一个信号及其直接目标，一个主宰性别决定开关的基因 这也控制着X染色体剂量补偿。然而，一个根本性的问题仍然存在： 信号以“全有或全无”的方式可重复地解释，以诱导可育的雄性或两性发育，从不 双性人发育？我们开创了使用机器学习神经网络来解决这个问题的新方法。 单分子和单细胞分辨率的问题。我们还建议对功能相互作用进行剖析 染色质修饰和染色体结构在调控基因表达中的作用 染色体的领地。线虫的X染色体剂量补偿是这项分析的典范：我们 最近发现，剂量补偿的X染色体具有(I)修饰的组蛋白水平升高 H4K20me1与常染色体相比；(Ii)独特的三维结构。两者都是由 剂量补偿复合体(DCC)。H4K20me1基因缺失扰乱3D架构并提升X基因 表情。在线虫DCC中，一个亚基是H4K20me2去甲基酶，五个亚基是同源物 凝集素亚基，紧凑并分解有丝分裂和减数分裂染色体。所有DCC亚基都是 由XX特异性亚基特异性地招募到两性体X染色体上，从而触发与顺式-DNA的结合 作用于X(Rex)的调控元件，使基因表达减少一半。DCC重塑了X的结构 利用其最高亲和力的rex位点建立结构域边界的拓扑结合结构域(TADS)。 尽管有这些知识，但剂量补偿机制背后的重要问题仍然存在。 哪些DCC亚基识别REX位点上富含X的基序，从而直接与X结合？DCC是如何监管的 RNA聚合酶II抑制基因表达？S控制H4K20me1的机制是什么 染色体结构，以及DCC介导的高阶结构如何影响基因表达？我们的 这些发现应该具有广泛的意义，因为(I)凝集素复合体从 细菌对人类的影响，(Ii)H4K20me1富含在雌性哺乳动物的非活性X上，(Iii)去甲基酶与 肿瘤进展，以及(Iv)H4K20me2去甲基酶调节线虫的生长、代谢和进入 静止的达尔状态。最后，我们将利用我们意想不到的发现，即Rex站点已经 线虫物种被30 MYR隔开，尽管强烈保存了 核心催化裂化设备。这种分歧提供了一个不同寻常的机会，可以研究协调一致的共同 X染色体和结合它们的蛋白质复合体上数百个靶点的进化变化。

PROJECT SUMMARY Studies are proposed to dissect one of the fundamental, binary development decisions that most metazoans make: their sex. The nematode C. elegans determines sex with remarkable precision by tallying X- chromosome number relative to the sets of autosomes (X:A signal): ratios of 1X:2A (0.5) and 2X:3A (0.67) signal male fate, while ratios of 3X:4A (0.75) and 2X:2A (1.0) signal hermaphrodite fate. We have discovered much about the nature and action of the X:A signal and its direct target, a master sex-determination-switch gene that also controls X-chromosome dosage compensation. However, a fundamental question remains: how is the signal interpreted reproducibly in an "all or none" manner to elicit fertile male or hermaphrodite development, never intersexual development? We pioneer new methods using machine learning neural networks to address this question with single-molecule and single-cell resolution. We also propose to dissect the functional interplay between chromatin modification and chromosome structure in regulating gene expression over vast chromosomal territories. X-chromosome dosage compensation in C. elegans is exemplary for this analysis: we found recently that dosage-compensated X chromosomes have (i) elevated levels of modified histone H4K20me1 compared to autosomes and (ii) a unique three-dimensional architecture. Both are imposed by the dosage compensation complex (DCC). Loss of H4K20me1 disrupts 3D architecture and elevates X gene expression. In the nematode DCC, one subunit is an H4K20me2 demethylase and five subunits are homologs of condensin subunits, which compact and resolve mitotic and meiotic chromosomes. All DCC subunits are recruited specifically to hermaphrodite X chromosomes by an XX-specific subunit that triggers binding to cis- acting regulatory elements on X (rex) to reduce gene expression by half. The DCC remodels the structure of X into topologically associating domains (TADs) using its highest affinity rex sites to establish domain boundaries. Despite this knowledge, important questions underlying the mechanisms of dosage compensation remain. What DCC subunits recognize the X-enriched motifs in rex sites to bind X directly? How does the DCC regulate RNA polymerase II to repress gene expression? What mechanisms underlie H4K20me1's control of chromosome structure, and how does DCC-mediated higher-order structure affect gene expression? Our findings should have broad implications, because (i) condensin complexes control chromosome structure from bacteria to man, (ii) H4K20me1 is enriched on the inactive X of female mammals, (iii) demethylases are linked to tumor progression, and (iv) the H4K20me2 demethylase modulates nematode growth, metabolism, and entry into the quiescent dauer state. Lastly, we will exploit our unexpected finding that rex sites have diverged across Caenorhabditis species separated by 30 MYR, retaining no functional overlap despite strong conservation of the core DCC machinery. This divergence provides an unusual opportunity to study the path for a concerted co- evolutionary change in hundreds of target sites across X chromosomes and the protein complexes that bind them.