Functional Analysis of programmed genome rearrangement

This proposal stems from our recent discovery that the sea lamprey (unlike other vertebrates) undergoes large-scale genome rearrangements during the normal course of its embryonic development. Programmed genome rearrangement (PGR) results in the highly reproducible elimination of ~20% of the lamprey's genome during the establishment of somatic cell lineages in the early embryo. Recent work has revealed that: 1) hundreds of gene-coding fragments are eliminated, 2) human homologs of lost genes generally possess "pluripotency" functions, 3) computational analysis of "next-gen" sequence datasets identify predicted recombination sites that mediate deletion events, which have been validated experimentally, indicating that PGR is mediated in part by recombination and 4) proteins expressed in the early embryo show sequence-specific binding to validated recombination sites. With the advent of extensive sequence resources for lamprey and methods for gene knockdown and transcript replacement, lamprey will continue to provide unique insights into the rearrangement biology of vertebrate genomes and the genetics of somatic vs. germline cell fate. As such, this research is broadly relevant to NIGMS research programs in genetics and developmental biology, particularly towards understanding the biology of genomes, chromosomes and epigenetics, vertebrate developmental genetics, stem cell biology, genetic mechanisms of DNA recombination/repair, and oncogenesis. The specific aims of this proposal will seek to further characterize lamprey PGR at the genetic level in order to deduce its molecular mechanisms and use this information to guide functional studies of the biological causes and consequences of genome rearrangement. Specific Aim 1 adds clarity to our understanding of genomic-wide patterns of gene loss, by developing a meiotic map for the lamprey genome and integrating this with existing somatic and germline genome assemblies. Specific Aim 2 dissects the function of genes that are deleted through PGR. Experiments performed under this aim will use morpholino mediated gene knockdowns to elucidate the function of somatically deleted genes in the embryonic lineages that retain germline-specific genes. Specific Aim 3 identifies genes that mediate PGR and characterizes the function of their human homologs. Experiments performed under this aim will use morpholino-mediated knockdown to elucidate the function of genes that bind recombination sites during PGR then use transcript replacement to test whether human homologs can partly or completely replace these functions. This will provide a direct assay for the capacity of identified human homologs to participate in somatic recombination. Critically, these approaches do not rely on pre-existing knowledge regarding the function of human genes and therefore hold great potential for the discovery of new gene functions. These studies are expected to provide unique insight into the biological function of human genes and yield information that might not be achievable outside of the context of PGR.