Our genomes are subject to a constant barrage of damage from reactive metabolites, environmental agents, or physiologic processes. A major form of physiologic damage is DNA double-strand breaks (DSBs) arising from transcription and replication. Developing lymphocytes also target DSBs to antigen receptor (AgR) loci as part of their assembly by V(D)J recombination. To maintain genomic stability, DSBs must be repaired with high fidelity, minimizing oncogenic alterations such as chromosomal deletions and translocations. The DSB response leads to extensive revision of flanking chromatin, including phosphorylation of the histone variant H2AX by the damage-sensing kinase ATM. Phosphorylated H2AX (?-H2AX) spreads for 100s of kb from a DSB. In non-cycling cells, the ?-H2AX domain serves as a chromatin-based platform to facilitate repair by the non-homologous end joining (NHEJ) machinery and, perhaps, as an adherent surface to hold broken chromosome ends together. Indeed, broken chromosomes are destabilized in cells deficient for ATM or H2AX, which have elevated levels of translocations. Thus, a deeper understanding of mechanisms that coordinate DSB repair and sequester lesions from other parts of the genome remains an important goal in cancer biology. In this regard, mechanistic links between DNA repair, transcription, and epigenetic landscapes around DSBs are beginning to emerge. A feature that may bridge these processes is the 3D conformation of chromatin flanking a DSB. However, the impact of DSBs on genome conformation and, conversely, the role of its reconfiguration in stabilizing DNA ends for repair, remain unexplored. Conformational mechanisms are likely important to generate compact platforms for repair complexes and to spatially restrict DSBs from other regions of the genome. A breakdown in these processes may destabilize unrepaired chromosome ends, allowing them to drift apart or to participate in translocations. The applicant has discovered that DSBs in precursor lymphocytes induce compaction of chromatin over 100s of kb flanking DSB sites in AgR loci, paralleling the spread of ?-H2AX. Borders of compacted ?-H2AX domains correspond with those of topologically associated domains (TADs), the architectural building blocks of chromosome structure. Launching from these discoveries, the overarching hypothesis of the project is that ?-H2AX domains are limited by inherent topological features around the DSB site, forming a spatially compact platform to stabilize association of chromosome ends and focus repair. Three aspects of the hypothesis will be studied: (i) how DSB location within a TAD affects the intensity and breadth of ?-H2AX domains, linking these features to translocation potential, (ii) how damage response factors mediate DSB-induced conformational changes and end stabilization, and (iii) how DSBs impact structural and regulatory loops that drive gene expression. Findings from this project will advance the field, providing new insights into how DSB responses integrate spatial, transcriptional, and chromatin-based mechanisms to sequester chromosome ends for efficient repair, minimizing their oncogenic potential.

Public Health Relevance

Damage to our genome, in the form of DNA double-strand breaks, must be recognized and efficiently repaired to avoid oncogenic aberrations, such as chromosomal translocations. The DNA break response includes extensive revisions of regional chromatin; however, the impact of spatial architecture surrounding damage sites on their subsequent repair remains unknown. This project will define how the three-dimensional conformation of our genome is altered by DNA breaks and, in turn, how these spatial reconfigurations aid in holding broken chromosome ends together for efficient repair, minimizing their potential for cancer-causing translocations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
7R01AI118852-05
Application #
9899620
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Liu, Qian
Project Start
2015-03-01
Project End
2021-02-28
Budget Start
2019-03-01
Budget End
2021-02-28
Support Year
5
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Ohio State University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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