Dr. Bonner received his Ph.D. from Harvard University in Biochemistry and Molecular Biology. He studied as a postdoctoral fellow at Oxford University and the MRC Laboratories in Cambridge, England. In 1974 he came to NIH and began his studies on histone functions as a Staff Fellow in the National Institute of Child Health and Human Development. Two years later he moved to NCI, continuing his studies on histone roles in chromatin in the Laboratory of Molecular Pharmacology.
The typical mammalian cell contains between two and four meters of DNA compacted to the 90-180 mm of 30 nm diameter fibers of interphase chromatin and further condensed to the 120 µm of 700 nm diameter arms of mitotic chromosomes. This compaction is accomplished by small basic proteins which complex with DNA to form nucleosomes--the structural and functional units of both interphase chromatin and mitotic chromosomes. The nucleosome comprises 145 bp DNA and eight small basic protein molecules called histones, two from each of the four core histone families, H4, H3, H2B and H2A. A minimum of another 20 bp of DNA stretches between nucleosomes complexed with the linker histone H1.
Einstein's maxim "Everything should be made as simple as possible, but not simpler." certainly has applied to chromatin. For years many researchers accepted this uniform nucleosome model as meeting Einstein's maxim, but with increasing cognizance of several complexities, most now recognize it as too simple. One intriguing complexity is the division of the histone H2A family into several subfamilies each with its own conserved sequence characteristics. In mammals the bulk H2A histones, meaning those species visible on an SDS gel of total cell extract, belong to the H2A1 subfamily while two other subfamilies, the H2AX and H2AZ each constitute about 10% of the total H2A (West and Bonner, 1980) . H2AX and H2AZ homologues are present throughout eucaryotic evolution. For example, budding yeast contains three H2A species; the two most plentiful are members of the H2AX subfamily, while the other is a member of the H2AZ subfamily.
These complexities of the H2A protein family suggested that histones may perform novel and unforeseen roles in chromatin. The human H2AX cDNA sequence revealed the C-terminal protein sequence KATQAS139QEY*, similar to the sequence KATKAS129QEL* reported for budding yeast H2A1 (* signifies the C-terminus). This finding demonstrated that higher eucaryotes contain an H2A species with a C-terminus very different from that of their bulk H2A species but at the same time strikingly homologous with that of bulk yeast H2A (Mannironi et al., 1989). We then found that mammalian H2AX is extensively phosphorylated within minutes after the introduction of DNA double-strand breaks (hereafter DSBs) into cells by ionizing radiation. We named this phosphorylated mammalian H2AX species γ-H2AX, localized its phosphorylation site to serine 139, and determined that hundreds to several thousand H2AX molecules become phosphorylated in the chromatin equivalent of at least 2 megabases of DNA per DSB (Rogakou et al., 1998).
To determine where γ-H2AX molecules are located in cells containing DNA DSBs, we generated an antibody specific to the C-terminus of human γ-H2AX (anti-γ). With anti-γ we demonstrated that the phosphorylation of the serine four residues from the C-terminus in response to ionizing radiation is conserved throughout eucaryotic evolution, occurring not only in mammalian species but also in the non-mammalian species, xenopus, fruit fly, and budding yeast. γ-H2AX appears as small punctate foci within the nuclei of mammalian cells exposed to ionizing radiation. The foci appear within one minute of irradiation and some foci grow for about 30 minutes to diameters of 0.5 µm. There do not appear to be large changes in the number of foci during the first 60 minutes after irradiation, but after that there are noticeable and continuing decreases in numbers of foci. The numbers of γ-H2AX foci obtained after subjecting cells to ionizing radiation is consistent with the predicted range of numbers of slowly-rejoining DNA double-strand breaks. IMR90 cells subjected to 0.6 Gray (an amount of radiation chosen because it is below the D0 (the dose permitting 37% clonal survival) for most mammalian cell types while inducing a countable number of γ-H2AX foci in a maximum projection) would be expected to contain numbers of DNA double-strand breaks consistent with a Poisson distribution peaking at 7 slowly-rejoining breaks. In such IMR90 cell cultures the number of γ-H2AX foci was measured to be 10.1 ± 3.9 after 15 minute recovery, 11.6 ± 5.3 after 30 minute, and 11.4 ± 6.1 after 60 minute recovery, values close to the predicted value of slowly-rejoining DNA double-strand breaks (Rogakou et al., 1999).
Using muntjac (a small deer) cells with giant chromosomes, we found that γ-H2AX foci also form on metaphase chromosomes with about the same kinetics as in interphase nuclei. On metaphase chromosomes the foci assume disk-like shapes which resemble chromosome bands. Strikingly, the vast majority of γ-H2AX disks appear in apparently intact chromosome arms even 90 minutes after irradiation during anaphase when the sister chromatids were being pulled apart. However, whenever abandoned chromosome arm fragments are present, one end is covered with a γ-H2AX focus.
In collaboration with Drs. Tanya Paull and Martin Gellert, we asked whether proteins involved in DNA DSB repair colocalize with γ-H2AX foci in vivo and whether foci formation by these proteins depend on γ-H2AX foci formation. We demonstrated the utility of targeted DNA DSBs with the LaserScissorsTM as a means of studying the relocalization of proteins to those lesions in vivo. RAD50, MRE11, and other proteins were found to localize to γ-H2AX foci. Wortmannin prevented γ-H2AX foci formation if added before irradiation, but not when added after irradiation. When γ-H2AX foci were absent, foci of other DNA repair proteins were also absent, suggesting that prior formation of γ-H2AX foci was necessary for subsequent formation of these other foci Paull et al., 2000).
In collaboration with Dr. Andre Nussenzweig's and Dr. Thomas Ried's laboratories in NCI, we asked whether or not γ-H2AX is involved in V(D)J recombination in vivo. We found that histone γ-H2AX foci are also found at sites of V(D)J (variable, diversity, joining) recombination induced DSBs. In developing thymocytes, γ-H2AX foci colocalize with the T cell receptor a locus in response to recombination activating gene (RAG) protein mediated V(D)J cleavage (Chen et al., 2000).
We had observed that testes from unirradiated mice contain considerable amounts of γ-H2AX by two dimension gel analysis and that γ-H2AX is present in testes sections. Thus we strongly suspected that γ-H2AX may be involved in meiotic recombination. Such was shown to be the case in great detail in collaboration with Dr. Paul Burgoyne (Division of Developmental Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London). During leptotene and zygotene, γ-H2AX antibody labels multiple chromatin domains that encompass those stretches of axial elements that have foci of the recombinase DMC1. These leptotene/zygotene γ-H2AX signals are Spo11-dependent and disappear as synapsis progresses. These data provide compelling evidence that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene, that the processing of the breaks is closely co-coordinated with synapsis and that the X non-PAR axis is not protected from these breaks. The intriguing and controversial point to come from this work was the finding spo11-/- mice, which lack the enzyme for making meiotic DNA DSBs, still showed γ-H2AX staining over the sex body (Mahadevaian et al., 2001).
Keywords: chromatin structure, ionizing radiation, DNA double-strand breaks, V(D)J recombination, meiotic recombination, histone H2AX, phosphorylation
Selected primary publications:
Last updated March 22, 2001