| Structural highlights
Function
H2AX_HUMAN Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.[1] [2] [3] [4]
Publication Abstract from PubMed
Histone H2AX phosphorylated at serine 139 (gamma-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While gamma-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to gamma-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of gamma-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize gamma-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect gamma-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision gamma-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.
A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.,Moeglin E, Desplancq D, Stoessel A, Massute C, Ranniger J, McEwen AG, Zeder-Lutz G, Oulad-Abdelghani M, Chiper M, Lafaye P, Di Ventura B, Didier P, Poterszman A, Weiss E Cancers (Basel). 2021 Jul 1;13(13):3317. doi: 10.3390/cancers13133317. PMID:34282773[5]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 2000 Jul 27-Aug 10;10(15):886-95. PMID:10959836
- ↑ Kobayashi J, Tauchi H, Sakamoto S, Nakamura A, Morishima K, Matsuura S, Kobayashi T, Tamai K, Tanimoto K, Komatsu K. NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr Biol. 2002 Oct 29;12(21):1846-51. PMID:12419185
- ↑ Stewart GS, Wang B, Bignell CR, Taylor AM, Elledge SJ. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature. 2003 Feb 27;421(6926):961-6. PMID:12607005 doi:10.1038/nature01446
- ↑ Lukas C, Melander F, Stucki M, Falck J, Bekker-Jensen S, Goldberg M, Lerenthal Y, Jackson SP, Bartek J, Lukas J. Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J. 2004 Jul 7;23(13):2674-83. Epub 2004 Jun 17. PMID:15201865 doi:10.1038/sj.emboj.7600269
- ↑ Moeglin E, Desplancq D, Stoessel A, Massute C, Ranniger J, McEwen AG, Zeder-Lutz G, Oulad-Abdelghani M, Chiper M, Lafaye P, Di Ventura B, Didier P, Poterszman A, Weiss E. A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress. Cancers (Basel). 2021 Jul 1;13(13):3317. PMID:34282773 doi:10.3390/cancers13133317
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