EAGER: Diversity of Animal Immunity and Somatic DNA Modifications in the Sea Urchin

免疫学家多年来一直在寻找脊椎动物中适应性免疫的起源，但收效甚微。了解这个复杂生物系统的起源和进化有助于了解其特征的细节，这将有助于开发解决人类疾病的最佳方法，例如靶向破坏癌细胞。海胆基因组具有一组独特的免疫基因，当检测到病原体时，这些基因被打开，并且编码大量的抗微生物（即，防御性）蛋白质具有相似的结构。在脊椎动物的适应性免疫系统中，抗体基因在免疫细胞中通过所谓的RAG酶“剪切和粘贴”其DNA序列而改变。RAG酶活性的结果是编码抗体的DNA序列的改组，从而在免疫防御中产生适应性变化，能够灵活地响应新的病原体。该项目的初步结果表明，海胆免疫基因家族发生了变化，这表明这些基因的DNA序列也可能通过“剪切和粘贴”机制发生变化。海胆也有RAG酶类似的脊椎动物。研究人员建议确定海胆免疫基因家族是否被“剪切和粘贴”事件改变，以及这是否由海胆RAG酶控制。如果是这样，他们还将确定RAG酶用于改变免疫基因家族的DNA靶点。确定一种机制，改变了海胆免疫基因家族的结构的“剪切和粘贴”的机制，这是目前只知道脊椎动物，预计将有显着的影响，我们的理解的进化起源，适应性免疫系统的功能。研究人员建议让科学家和非科学家在地方，国家和国际会议上讨论免疫系统如何发挥作用。将确定少数群体和代表性不足的群体并邀请他们参加。他们将为当地小学生提供一系列动手示范，教他们有关海洋无脊椎动物、它们的生态以及如何将它们用于科学研究。紫海胆中的Sp185/333基因家族有多达60个基因，这些基因很小，紧密聚集，显示出非凡的序列多样性。Sp185/333基因由吞噬细胞表达以响应免疫攻击，并且单个吞噬细胞表达单个Sp185/333基因，推断来自该家族的表达的显著限制。免疫攻击的体腔细胞与未攻击的对照相比显示出新的基因大小，其还显示出不同体腔细胞样品之间以及与其他成体组织相比的基因大小比率的变化。研究人员推测，Sp185/333基因家族的体细胞变化可能是限制单个吞噬细胞中Sp185/333基因表达的基础。他们建议使用全基因组扩增、片段（=扩增子）大小分析和扩增子测序来表征单个吞噬细胞中Sp185/333基因家族的结构。FISH将用于检测Sp185/333基因家族结构的可能大规模变化。此外，他们建议鉴定可能参与Sp185/333 DNA修饰的酶，他们将专注于RAG 1/2同系物加上TdT和Artemis，所有这些都是基于RNAseq数据表达的。SpRAG-2L的抗体将与ChIP一起用于从吞噬细胞捕获染色质，以鉴定SpRAG-2L可能结合的靶序列。将使用ChIP鉴定的染色质序列作为诱饵，用海胆吞噬细胞的蛋白裂解物进行凝胶迁移，以确认结果。结合蛋白质将确定MS。结果预计将提供海胆吞噬细胞的免疫多样化，使他们能够跟上对病原体的军备竞赛的信息。这种机制可能与脊椎动物所采用的机制相似，预计将拓宽或改变我们对动物免疫进化、适应性免疫起源的认识，并将为无脊椎动物免疫学家带来新的研究方向。

Immunologists have searched for the origins of adaptive immunity in vertebrates for years with only limited success. Knowledge of the origins and evolution of this complex biological system can aid in understanding the details of its characteristics, which will enable development of optimal approaches for solving human diseases such as the targeted destruction of cancer cells. The sea urchin genome has a unique set of immune genes that are turned on when pathogens are detected, and which encode a large array of anti-microbial (i.e., defensive) proteins that have similar structures. In the vertebrate adaptive immune system, antibody genes are changed in immune cells by "cutting and pasting" their DNA sequences by so-called RAG enzymes. The consequence of the RAG enzyme activity is a shuffling of the DNA sequences that code for the antibodies, thereby producing the adaptive variation in immune defense that is capable of responding flexibly to new pathogens. The preliminary results presented in this project show that there are changes to the sea urchin immune gene family suggesting that the DNA sequences for these genes may also change by a "cut and paste" mechanism. Sea urchins also have RAG enzymes similar to those of vertebrates. The investigators propose to determine whether the sea urchin immune gene family is altered by "cutting and pasting" events and if this is controlled by the sea urchin RAG enzymes. If so, they will also identify the DNA targets that the RAG enzymes use to change the immune gene family. Identifying a mechanism that alters the structure of a sea urchin immune gene family by the "cut and paste" mechanism, which is currently only known for vertebrates, is expected to have a significant impact on our understanding of the evolutionary origin of, and functioning of adaptive immune systems. The investigators propose to engage scientists and non-scientists in discussions of how immune systems function at local, national and international meetings. Minorities and underrepresented groups will be identified and invited to participate. They will present a series of hands-on demonstrations for local grade school children to teach them about marine invertebrates, their ecology and how they can be used for scientific research. The Sp185/333 gene family in the purple sea urchin has up to 60 genes, which are small, tightly clustered, and show extraordinary sequence diversity. The Sp185/333 genes are expressed by phagocytes in response to immune challenge, and single phagocytes express a single Sp185/333 gene, inferring significant restriction of expression from the family. Immune challenged coelomocytes show a new gene size compared to non-challenged controls, which also show changes in the ratios of gene sizes among different coelomocyte samples and compared to other adult tissues. The investigators hypothesize that somatic changes to the Sp185/333 gene family may be the basis for restricting the Sp185/333 gene expression in individual phagocytes. They propose to characterize the structure of the Sp185/333 gene family in single phagocytes using whole genome amplification, fragment (= amplicon) size analysis, and amplicon sequencing. FISH will be used to detect possible large scale changes to the Sp185/333 gene family structure. In addition, they propose to identify enzymes that may be involved in the Sp185/333 DNA modifications and they will focus on RAG1/2 homologues plus TdT and Artemis, all of which are expressed based on RNAseq data. Antibodies to SpRAG-2L will be used with ChIP to capture chromatin from phagocytes to identify target sequences to which SpRAG2L may bind. Results will be confirmed using chromatin sequences identified by ChIP as baits for gel shifts with protein lysates from sea urchin phagocytes. Bound proteins will be identified by MS. Results are expected to provide information on immune diversification in sea urchin phagocytes that enables them to keep pace in the arms race against their pathogens. The mechanism(s) may be similar to that employed by vertebrates, and are expected to broaden or perhaps alter our thinking about the evolution of animal immunity, the origins of adaptive immunity, and will lead to new lines of investigation by invertebrate immunologists.