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

George Washington University, Washington DC

Investigators

Abstract

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.