Our long-term objective is to help decipher the mechanism of DNA double-strand break (DSB) repair, a process indispensable in maintaining genomic stability in all organisms, and an important barrier to cancer in higher eukaryotes. Reversible site-specific post-translational modifications of histone and non-histone proteins are essential for the DNA repair machinery to assemble at sites of DNA DSBs and to the concomitant checkpoint activation leading to cell cycle arrest or apoptosis, but how these modifications contribute to the DNA damage response is poorly understood. Our proposed research focuses on understanding the role of lysine methylation in DNA DSB and cell cycle checkpoint signaling. We will probe several molecular interactions driven by the methylation of histone H4 at lysine 20 (H4-K20) and p53 at lysine 370 (p53-K370), and their possible synergistic coupling to other post-translational modifications (e.g. phosphorylated histone H2AX). In particular, we will study the interactions of methylated histone H4-K20 and methylated p53-K370 with human proteins 53BP1, JMJD2A and PHF20. 53BP1 is a key transducer of the cell response to DNA DSBs that participates in the assembly of DNA repair proteins and in checkpoint activation regulated by p53. JMJD2A is a histone demethylase whose function is linked to p53-dependent DNA damage-induced apoptosis. PHF20 is a component of the MLL1/MOF histone acetyltransferase complex that acetylates histone H4 in a p53 dependent manner in connection to DNA damage. For each study, we will apply a combination of biophysical experiments centered on nuclear magnetic resonance (NMR) spectroscopy and crystallographic three-dimensional (3D) structure determination of selected domains in complex with their associated targets.
In Aim1, we will test the hypothesis that 53BP1 simultaneously recognizes methylated H4-K20 and phosphorylated histone H2AX using tandem tudor and tandem BRCT domains, respectively. This dual binding mode would explain how 53BP1 is recruited to DNA damage sites.
In Aims 2 and 3, we will test the hypotheses that methylation of p53 at lysine 370 triggers a tight interaction of p53 with 53BP1 and JMJD2A, preventing p53 from binding DNA. This would provide a mechanism for the known inhibition of p53 transcriptional activity by methylation of lysine 370.
In Aim 4, we have identified a new motif in PHF20 that we call disulfide cross-linked tudor dimer. This tudor domain is found in tandem with an MBT domain. We will characterize the tudor, MBT and tandem MBT-tudor domains of PHF20 in complex with methylated H4-K20 and p53-K370 peptides. Our structural and interaction studies at the atomic level will be the basis for informed design of in vivo experiments that will elevate our knowledge of DNA DSB signaling and repair. Alteration of histone H4 methylation at Lys20 is a hallmark of human tumor cells and mutation of p53 occurs in about 50% of human cancers. In the long term, our studies involving methylated histone H4 and p53 may help control the DNA damage response therapeutically for the prevention and treatment of cancer.

Public Health Relevance

The proposed studies will contribute to the elucidation of fundamental cellular processes involved in detecting and repairing DNA double-strand breaks, one of the most harmful types of DNA damage. The work has relevance to public health because by understanding the mechanisms of these important processes, we may be able to find ways to prevent and treat human malignancies, particularly cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Pelroy, Richard
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Mayo Clinic, Rochester
United States
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Botuyan, Maria Victoria; Cui, Gaofeng; Drané, Pascal et al. (2018) Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein. Nat Struct Mol Biol 25:591-600
Cui, Gaofeng; Botuyan, Maria Victoria; Mer, Georges (2018) Structural Basis for the Interaction of Mutasome Assembly Factor REV1 with Ubiquitin. J Mol Biol 430:2042-2050
Drané, Pascal; Brault, Marie-Eve; Cui, Gaofeng et al. (2017) TIRR regulates 53BP1 by masking its histone methyl-lysine binding function. Nature 543:211-216
Mer, Georges; Botuyan, Maria Victoria (2017) A New BRCT Binding Mode in TopBP1-BLM Helicase Interaction. Structure 25:1471-1472
Klein, Christopher J; Wu, Yanhong; Jentoft, Mark E et al. (2017) Genomic analysis reveals frequent TRAF7 mutations in intraneural perineuriomas. Ann Neurol 81:316-321
Hu, Qi; Botuyan, Maria Victoria; Cui, Gaofeng et al. (2017) Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18. Mol Cell 66:473-487.e9
Valieva, Maria E; Gerasimova, Nadezhda S; Kudryashova, Kseniya S et al. (2017) Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy. Cancers (Basel) 9:
Klein, Brianna J; Wang, Xiaoyan; Cui, Gaofeng et al. (2016) PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation. Cell Rep 17:1158-1170
Kanakkanthara, Arun; Jeganathan, Karthik B; Limzerwala, Jazeel F et al. (2016) Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation. Science 353:1549-1552
Perfetti, Michael T; Baughman, Brandi M; Dickson, Bradley M et al. (2015) Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1. ACS Chem Biol 10:1072-81

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