The epigenetic basis of cytoplasmic incompatibility

沃尔巴克氏菌是一种细胞质遗传的细菌，通过一种被称为细胞质不亲和性(CI)的宿主操作在数千种动物中传播。CI是一种交配后的不亲和性，使感染沃尔巴克氏菌的雄性与未感染的雌性不相容。然而，受感染的雄性和受感染的雌性之间的交配是相容的，从而使受感染的雌性具有健康优势。支持CI的分子机制近20年来一直令生物学家难以捉摸，尽管在宿主-细菌相互作用、遗传冲突、动物物种形成和病媒控制策略的进化中应用明显，以遏制人类疾病。本项目的目标是探索脑梗塞分子机制的新假说。我们认为CI是由一种感染性的表观遗传学改变引起的，在这种改变中，沃尔巴克氏菌通过操纵DNA甲基化来加密精子和卵子。这一假说基于三个关键点：(I)首先，由于沃尔巴克氏菌在精子发生过程中被从细胞质废袋中的精子中剥离，CI必须涉及到对感染沃尔巴克氏菌的男性睾丸中的精子进行“修饰”。受感染女性的卵子也必须能够“拯救”沃尔巴克氏菌--精子改造。这些针对性别的加密是表观遗传现象的特征。(Ii)第二，CI杂交导致的胚胎无活力发生在受精卵的第一次缓和分裂期间和之前，表观遗传现象被认为是重要的。(Iii)第三，我们的初步证据表明，用DNA甲基化小分子抑制剂(5-azacytidine)治疗的受感染的黑腹果蝇，当只用一种性别的药物治疗时，表现出CI。然而，在未感染的对照杂交中没有观察到这种效应。此外，对感染的男性和女性的治疗导致了亲和性的恢复，这表明母亲和父亲基因组之间的DNA甲基化水平的差异导致了CI。为了确定沃尔巴克氏菌是否会改变DNA甲基化导致CI，我们将在80倍的覆盖率下对整个基因组进行亚硫酸氢盐测序，以比较感染沃尔巴克氏菌和未感染的果蝇的DNA甲基化模式。由于DNA甲基化和基因表达受到严格调控，我们接下来将用来自相同样本的RNA-SEQ对整个转录组进行测序，以确定感染和未感染沃尔巴克氏菌的果蝇之间基因表达的差异，这些差异可以追溯到基于感染状态的DNA甲基化的差异。最后，我们将确定沃尔巴克氏菌的修饰是否需要保守的动物DNA甲基化和去甲基化基因。这项研究将首次确定沃尔巴克氏杆菌是否会引起感染性表观遗传学改变，从而导致脑梗塞。

Wolbachia are cytoplasmically inherited bacteria that have spread through thousands of animal species by a host manipulation called cytoplasmic incompatibility (CI). CI is a postmating incompatibility that renders Wolbachia-infected males incompatible with uninfected females. However, matings between infected males and infected females are compatible, thereby imparting a fitness advantage to infected females. The molecular mechanism that underpins CI has evaded biologists for almost two decades, despite obvious, applications to the evolution of host-bacterial interactions, genetic conflict, animal speciation, and vector control strategies to curb human diseases. The goal of this project is to investigate a new hypothesis for the molecular mechanism of CI. We propose that CI results from an infectious epigenetic alteration in which Wolbachia encrypt the sperm and egg through a manipulation of DNA methylation. This hypothesis is based on three key points: (i) First, since Wolbachia are stripped from the sperm in the cytoplasmic waste bag during spermatogenesis, CI must involve a "modification" of the sperm in the testes of a Wolbachia-infected male. The eggs of an infected female must also be able to "rescue" the Wolbachia-sperm modification. These gender-specific encryptions are hallmarks of epigenetic phenomena. (ii) Second, the embryonic inviability that results from a CI cross occurs during and preceding the first mitiotic division of the fertilized egg, where epigenetic phenomena are known to be important. (iii) Third, our preliminary evidence shows that infected Drosophila melanogaster flies treated with a small molecule inhibitor of DNA methylation (5-Azacytidine) exhibit CI when only one gender is treated with the drug. This effect, however, is not observed in uninfected, control crosses. Furthermore, treatment of both infected males and females leads to a restoration of compatibility, suggesting that differences in DNA methylation levels between the maternal and paternal genome lead to CI. To determine if Wolbachia alters DNA methylation to cause CI, we will bisulfite sequence the entire genome at 80X coverage to compare DNA methylation patterns in Wolbachia-infected and uninfected flies. Since DNA methylation and gene expression are tightly regulated, we will next sequence the whole transcriptome with RNA-seq from the same samples to identify differences in gene expression between Wolbachia-infected and uninfected flies that can be correlated back to the differences in DNA methylation based on infection status. Finally, we will determine if conserved, animal DNA methylation and demethylation genes are required for the modification by Wolbachia. This research will determine, for the first time, if Wolbachia induces an infectious epigenetic alteration to cause CI.