Although repeat sequences are found at unstable genomic regions implicated in cancer and genetic diseases, the mechanisms by which repeats contribute to genomic instability are poorly understood. The emerging view is that repeats form tracts of non-canonical chromatin structures that impair chromatin associated activities, such as DNA replication, transcription, and repair.  However, experimental approaches have relied on artificial global inhibition of the DNA replication machinery, which induces breaks at thousands of replication forks, often resulting in cell cycle arrest and loss of viability.  Binding of a sequence-specific DNA binding protein at a single cluster of repeats is a particularly valuable system for inducing site-specific repeat instability.  Thus, we are excited about leveraging EBNA1 binding at a single cluster of EBV-like repeats to define how protein binding to a tract of repeat sequences leads to breakage.

Cytogenetic studies have long established that breakage-prone sites in the genome correlate with recurrent breakpoints in cancer. However, beyond correlative data, there is limited direct experimental evidence of the role of a single break in initiating genomic abnormalities in disease. We have shown that EBNA1-induced breakage at chromosome 11q23 produces chromosomal fragments that undergo continued cycles of mis-segregation and micronucleation, which would lead to further breakage and error-prone reassembly. We will now identify breakage-induced sequence rearrangements at the initial site of breakage, along the rest of chromosome 11, and elsewhere in the genome, with the larger goal of uncovering a previously missing link between EBV and human diseases characterized with abnormalities of chromosome 11.

How does lifelong latent infection with EBV remain harmless in most of the human population, yet contribute to the development of cancer in some individuals? We showed that while latently infected cell lines producing baseline levels of EBNA1 do not exhibit signs of rampant chromosome 11 abnormalities, a doubling of the EBNA1 levels was sufficient to trigger chromosomal breakage. These results suggest that a threshold level of unstable EBNA1 binding-induced structures formed at the repeats is required to trigger breakage.  We will now investigate two physiological factors critical for triggering breakage: genetic variation of the repeat sequences in the human population, and regulation of nuclear levels of EBNA1 available for binding. 

Since their discovery, repeat sequences in our genome have been viewed as remnants of ancient viral infection.  Our discovery of the human repeats that share sequence homology with Epstein Barr Virus repeats provides an exciting opportunity to investigate their origin and potential function, fundamental to our understanding of the relationship between human and viruses.  Fascinatingly, these virus-like repeats are prone to breakage, yet conserved amongst primates. Thus, we are excited about using cellular assays to test their role in latent viral infection, and/or host genomic structure and function.