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Identifying Functional Proteins at DNA Breaks with Quantitative Proteomics in Primary Lymphocytes

Final Report Summary - DDR IN LYMPHOCYTES (Identifying Functional Proteins at DNA Breaks with Quantitative Proteomics in Primary Lymphocytes)

Chromatin accessibility is of key importance to understand how the DNA/chromatin template senses and amplifies the DNA damage signal that emanates from DNA double-strand breaks (DSBs) and how it facilitates DNA repair in its native context. DSBs in our genome that are not repaired properly can lead to immunodeficiency, various developmental and neurological diseases, and are a major driver for genomic instability and tumorigenesis. Following DSB detection, post-translational modifications of the histone protein components of chromatin and other proteins accumulate and spread away from the break site to generate a specialized chromatin domain with DNA-damagespecific characteristics; this cytologically discernible DNA damage response (DDR) promotes the preservation of genetic material and can result in cell-cycle arrest, DNA repair, or apoptosis. Phosphorylation at serine 139 of the H2AX histone variant of H2A (γH2AX) is the most striking and clear example of how a particular chromatin modification promotes genome stability. Mice deficient in H2AX accumulate spontaneous DSBs and rapidly develop tumors when cell-cycle checkpoints are compromised. However, it is poorly understood how chromatin-associated factors coordinate and care for these mutagenic events to suppress genomic instability and lymphoid cancer.
Here we develop a novel approach combining chromatin immunoprecipitation and mouse genetics with mass spectrometry-based label-free quantitative proteomics. To analyze chromatin on a proteomic scale, we optimized a biochemical method called chromatin enrichment for proteomics (ChEP) to enrich for cross-linked chromatin fragments specifically associating with γH2AX, a hallmark of DSBs. Using γ-irradiated lymphocytes from wild-type and H2AX-/- mice, this method allowed us to determine protein landscapes at DSBs with unprecedented resolution and accuracy.
Interestingly, we identified novel chromatin-associated proteins with, thus far, uncharacterized roles in the DNA damage response. Using cell biological methods, we confirmed that numerous identified proteins undergo prominent enrichment at DSB sites. To begin understanding the potential physiological roles of the newly identified factors, we performed targeted genetic screening in human cells and have observed increased sensitivity in response to genotoxic stress, suggesting direct functions for some of these proteins in maintaining genome stability. These combined unbiased proteomic and focused cell-based studies has deepen our understanding of the role of chromatin in the DDR and DSB repair and may provide relevant targets for rationale design of therapeutic strategies for cancer or immunodeficiency disease.