Genomic Conflict Resolution: Establishment of a new model for unconstrained germline selection

PROJECT SUMMARY:
Genes are regulated differently throughout the body, and mutations that may benefit one kind of cell may conflict
with the regulatory needs of other tissues. When mutations occur in reproductive cells, particularly ones that
provide a “selfish” advantage, these variations are transmitted to all of the offspring’s cells, representing a form
of germline selection that often conflicts with genetic processes of somatic tissues in resulting offspring. These
germline-somatic conflicts often serve as the primary driver of many spontaneous congenital disorders, and
many more variants of unknown significance (VUS) may be similarly influenced by poorly understood
evolutionary pressures that are uniquely subjected to the germline. Current approaches to study germline
selection are limited in their ability to study the natural emergence of germline mutations in a causal and iterative
fashion. This proposal addresses the need for a generalizable and comprehensive model by establishing a novel
model of germline selection using the germline restricted chromosome (GRC) of songbirds. This unique genomic
element contains reproductively-relevant gene duplications that have subtly diverged over evolution, offering a
promising avenue to explore germline selection mechanisms that are not restricted by somatic interference
across generations, mirroring the spontaneous processes impacting selfish DNM transmission and germline
selective congenital disorders. The project seeks to establish the zebra finch as a unique and comparative model
of germline-somatic conflict through three key aims across a mentored and independent research phase. In Aim
1, the candidate will enrich for GRC DNA samples by flow cytometry of mosaic testicular tissue for long-read
sequencing that will derive a high-quality GRC assembly. This assembly will then be used to identify sequence
differences between duplicate regions of the GRC and the rest of the genome. Aim 2 will utilize a comprehensive
collection of single-nuclei multiomic (RNA + ATAC) germline datasets across the vertebrate phylogeny to identify
the impact of GRC gene paralog sequence variations uniquely modifying gene regulatory networks in the zebra
finch germline, which will be further refined during the independent phase of the award using complementary
transcriptional and histological analyses. Aim 3 will apply CRISPR gene editing tools in zebra finch germline cells
to clarify the functional significance of GRC genes that maintain and enhance the evolutionary fitness of the
zebra finch germline, leveraging the GRC assembly and network analyses developed in the mentored phase.
This proposal will benefit from the candidate's expertise in avian genetics and biotechnology, along with training
in genome assembly and alignment, gene co-expression and cis-regulatory network analyses, and a foundation
in germline genetic processes across vertebrates. The work executed in this proposal will establish the zebra
finch GRC as a uniquely informative and broadly applicable model system to understand germline selection
mechanisms. Ultimately, this proposal will advance our understanding of genetic regulation, evolutionary
processes, and the assessment of complex genomic elements that will broadly impact biomedical research.

Grant Number: 1K99HG014014-01
NIH Institute/Center: NIH
Principal Investigator: Matthew Biegler