Immunophylogenetic aspects of a gorgonian coral

One goal of comparative immunology is to derive inferences about evolutionary pathways in the development of immune-defense systems. Almost 700 million years ago, a major divergence occurred in the phylogeny of animals, spitting all descendants into either the protostome or deuterostome (includes vertebrates) lineages. Genes have evolved independently along these lineages for that amount of time. Cnidarians originated before that divergence event, and can hold clues as to which immune response genes are homologous to both lineages. This work uses the gorgonian coral, Swiftia exserta, for two major reasons: (1) because of their phylogenetic position, corals are an important animal model in studies concerning the phylogeny of immune-response genes, and (2) nothing is known about the genes controlling immunocompetence in corals. The work described here has important implications in both innate and adaptive immunity. ^ The vertebrate complement system is a major component of innate immunity. C3 is a critical component of the three pathways of complement. Because of its opsonic properties, a C3-like protein is expected to have evolved early. However, currently available data suggests that complement-like components are unique to the deuterostome lineage. This work describes the cloning and characterization of a C3-like gene from S. exserta. The deduced polypeptide sequence reveals conservation of multiple, functionally critical, sites while sharing physiochemical and structural properties with the complement components C3/C4/C5. ^ Antigen processing, via intracellular enzymatic proteasomes, is a major requirement of vertebrate adaptive immunity. These organelles have a catalytic core, through which pass intracellular proteins for degradation into peptides presentable to the immune system. LMP 7 is one component of the paralogous “immuno-proteasome”. LMP 7 is a paralog of the ubiquitous LMP X, but is restricted to vertebrates. While LMP 7 is absent in the coral, this work describes a coral LMP X gene. Phylogenetic analyses, along with hydropathy profiling of a critical portion of the invertebrate and vertebrate paralogous genes, suggests that some invertebrates have two diverging LMP X genes. In some cases, one LMP X protein shares characteristics with vertebrate LMP 7. This work presents new evidence for how the LMP X and 7 genes evolved. ^

Reverse Engineering of Modified Genes by Bayesian Network Analysis Defines Molecular Determinants Critical to the Development of Glioblastoma

In this study we have identified key genes that are critical in development of astrocytic tumors. Meta-analysis of microarray studies which compared normal tissue to astrocytoma revealed a set of 646 differentially expressed genes in the majority of astrocytoma. Reverse engineering of these 646 genes using Bayesian network analysis produced a gene network for each grade of astrocytoma (Grade I–IV), and ‘key genes’ within each grade were identified. Genes found to be most influential to development of the highest grade of astrocytoma, Glioblastoma multiforme were: COL4A1, EGFR, BTF3, MPP2, RAB31, CDK4, CD99, ANXA2, TOP2A, and SERBP1. All of these genes were up-regulated, except MPP2 (down regulated). These 10 genes were able to predict tumor status with 96–100% confidence when using logistic regression, cross validation, and the support vector machine analysis. Markov genes interact with NFkβ, ERK, MAPK, VEGF, growth hormone and collagen to produce a network whose top biological functions are cancer, neurological disease, and cellular movement. Three of the 10 genes - EGFR, COL4A1, and CDK4, in particular, seemed to be potential ‘hubs of activity’. Modified expression of these 10 Markov Blanket genes increases lifetime risk of developing glioblastoma compared to the normal population. The glioblastoma risk estimates were dramatically increased with joint effects of 4 or more than 4 Markov Blanket genes. Joint interaction effects of 4, 5, 6, 7, 8, 9 or 10 Markov Blanket genes produced 9, 13, 20.9, 26.7, 52.8, 53.2, 78.1 or 85.9%, respectively, increase in lifetime risk of developing glioblastoma compared to normal population. In summary, it appears that modified expression of several ‘key genes’ may be required for the development of glioblastoma. Further studies are needed to validate these ‘key genes’ as useful tools for early detection and novel therapeutic options for these tumors.

Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.