Defining the Role of DNA Rereplication of Chromosome 21 in the Development and Pathophysiology of Down Syndrome

PROJECT SUMMARY.

Despite knowing for more than 60-years that three copies of chromosome 21 (chr21, T21) is the genetic cause of Down syndrome (DS), we still know relatively little about how T21 drives the vast majority of DS pathophysiology.

Over the last several decades, alterations in the response to diverse environmental stress conditions, such as oxidative stress, the unfolded protein response, the integrated stress response, and immune signaling, have been identified in T21 cells and mouse models of DS.

Indeed, we have shown that the interferon (IFN) response is constitutively active in T21 cells, leading to an interferonopathy-like state of global immune dysregulation, likely due to the presence of four IFN receptors (IFNRs) in a single locus on chr21.

Recently, we identified the IFNR locus as a target of directed transient site-specific gene amplification (TSSG) in response to viral infection.

Importantly, we found that while the IFNR cluster is subject to TSSG in healthy cells, this process is impaired in T21 cells.

These TSSGs arise from DNA rereplication events that are normally regulated by epigenetic changes to chromatin structure.

Our prior work determined that DNA rereplication and TSSG are part of cellular responses to myriad environmental stress, including hypoxia, UPR, and heavy metal exposure, implicating dysregulation of DNA rereplication as a potential common element in the altered stress responses of T21 cells.

This project will test the transformative hypothesis that dysregulated DNA rereplication in response to cellular stress contributes to the development of T21 and subsequent pathophysiology of DS. The potential impact of this hypothesis is profound.

Not only could the dysregulation of IFNR TSSG affect the response to immune activation and pathogen response, it is entirely possible that other stress response- mediated TSSGs are also impaired, with subsequent effects on systemic biology.

Furthermore, recent studies have shown that extrachromosomal DNA can lead to missegregation of chromosomes, leading to our hypothesis that virally-induced DNA rereplication of the IFNR locus during gamete development could promote missegregation of chr21 leading to T21.

Given the novelty of our observation and hypothesis, this proposal seeks to answer a number of questions regarding TSSG in T21 including: 1) How do T21 cells suppress TSSG of the IFNR locus? 2) Do T21 cells fail to initiate rereplication at other stress-induced TSSGs? 3) Do T21 cells have a different DNA rereplication program? and 4) How might amplification of the IFNR locus contribute to missegregation events that lead to T21?

Using a combination of cell culture models from humans and mice and a novel mouse in vitro fertilization model, we will comprehensively determine the extent of dysregulation of DNA rereplication in T21 and how DNA rereplication may contribute to the development of T21 and pathophysiology of DS.