Chipping away at gamma-H2AX foci

The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form gamma-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure gamma-H2AX densities along chromosomal DNA strands broken in G(1) phase mouse lymphocytes. Our experiments revealed that (1) gamma-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous if studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel gamma-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites

HECTD3 mediates an HSP90-dependent degradation pathway for protein kinase clients

Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre–B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igκ DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-α/δ locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs

Regulation of H2AX phosphorylation density around a defined double stranded DNA break in chromatin

One of the earliest events in cellular response to a nascent double stranded DNA break (DSB) is local induction of the phosphorylated histone H2AX (γH2AX). Phosphorylated by ATM and DNA-PK, γH2AX leads to retention of DNA damage response factors and facilitates the high fidelity DNA repair. In vivo, H2AX functions as a haploinsufficient tumor suppressor supressing genomic instability, thus the density of H2AX in chromatin appears to affect its function. Although γH2AX is localized in the region of chromatin harboring the break, localization with respect to the break site has remained largely unknown. In order to better understand the function of γH2AX and to address the question of its localization, I developed a system in which upon pharmacological stimulation, I can activate the Rag recombinase and induce double stranded breaks at the immunoglobulin kappa locus. Through a newly developed assisted chromatin immunoprecipitation assay, I found that γH2AX extends 400–500kb to one side of the break in chromatin. High density of γH2AX was found predominantly proximally to the break and was dependent on ATM anchoring to chromatin through MDC1. In the absence of either factor, γH2AX density was reduced five fold and the remaining γH2AX was induced solely through DNA-PK. In contrast, distal phosphorylation was mediated exclusively by ATM and was independent of MDC1. Inhibition of ATM-mediated phosphorylation of H2AX or the dephosphorylation of γH2AX through protein phosphatase 2A led to a marked decrease in γH2AX density. Notably, the levels of H2AX in chromatin also considerably affected the γH2AX density since 2–3 fold decrease in H2AX chromatin density results in a 10-fold decrease in γH2AX density. These data argue that there is a defined profile of γH2AX density around an unrepaired DSB. The maximal density of γH2AX depends on anchoring of ATM to chromatin and availability of the H2AX substrate for phosphorylation. Maximal distance is predominantly regulated by the nucleoplasmic pool of ATM and is independent of MDC1 anchoring. Notably, the observed phosphorylation profile is the result of a dynamic equilibrium between γH2AX induction and removal, potentially through γH2AX→H2AX histone exchange, creating a structure that is highly responsive to changes in ATM activity and potentially the repair status of the break

Regulation of H2AX phosphorylation density around a defined double stranded DNA break in chromatin

One of the earliest events in cellular response to a nascent double stranded DNA break (DSB) is local induction of the phosphorylated histone H2AX (γH2AX). Phosphorylated by ATM and DNA-PK, γH2AX leads to retention of DNA damage response factors and facilitates the high fidelity DNA repair. In vivo, H2AX functions as a haploinsufficient tumor suppressor supressing genomic instability, thus the density of H2AX in chromatin appears to affect its function. Although γH2AX is localized in the region of chromatin harboring the break, localization with respect to the break site has remained largely unknown. In order to better understand the function of γH2AX and to address the question of its localization, I developed a system in which upon pharmacological stimulation, I can activate the Rag recombinase and induce double stranded breaks at the immunoglobulin kappa locus. Through a newly developed assisted chromatin immunoprecipitation assay, I found that γH2AX extends 400–500kb to one side of the break in chromatin. High density of γH2AX was found predominantly proximally to the break and was dependent on ATM anchoring to chromatin through MDC1. In the absence of either factor, γH2AX density was reduced five fold and the remaining γH2AX was induced solely through DNA-PK. In contrast, distal phosphorylation was mediated exclusively by ATM and was independent of MDC1. Inhibition of ATM-mediated phosphorylation of H2AX or the dephosphorylation of γH2AX through protein phosphatase 2A led to a marked decrease in γH2AX density. Notably, the levels of H2AX in chromatin also considerably affected the γH2AX density since 2–3 fold decrease in H2AX chromatin density results in a 10-fold decrease in γH2AX density. These data argue that there is a defined profile of γH2AX density around an unrepaired DSB. The maximal density of γH2AX depends on anchoring of ATM to chromatin and availability of the H2AX substrate for phosphorylation. Maximal distance is predominantly regulated by the nucleoplasmic pool of ATM and is independent of MDC1 anchoring. Notably, the observed phosphorylation profile is the result of a dynamic equilibrium between γH2AX induction and removal, potentially through γH2AX→H2AX histone exchange, creating a structure that is highly responsive to changes in ATM activity and potentially the repair status of the break

ATM prevents unattended DNA double strand breaks on site and in generations to come

Ataxia telangiectasia (A-T) is a disorder characterized by cerebellar degeneration, immunodeficiency, genomic instability and genetic predisposition to lymphoid malignancies with translocations involving antigen receptor loci. The Ataxia Telangiectasia Mutated gene encodes the ATM kinase, a central transducer of DNA damage signals. Until recently, the etiology of the lymphoid phenotype in A-T patients and the mechanisms by which ATM ensures normal repair of DNA double strand break (DSB) intermediates during antigen receptor diversification reactions remained poorly understood. Last year, Bredemeyer et al. (Nature 2006; 442:466-70) demonstrated that ATM stabilizes chromosomal V(D)J recombination DSB intermediates, facilitates DNA end joining and prevents broken DNA ends from participating in chromosome deletions, inversions and translocations. A more recent study by Callen et al. (Cell 2007; 130:63-75) highlighted the importance of ATM-mediated checkpoints in blocking the long-term persistence and transmission of un-repaired DSBs in developing lymphocytes. Collectively, these results have provided complementary mechanistic insights into ATM functions in V(D)J recombination that can account for the lymphoid tumor-prone phenotype associated with A-

Novel synthetic lethality screening method identifies TIP60-dependent radiation sensitivity in the absence of BAF180

In recent years, research into synthetic lethality and how it can be exploited in cancer treatments has emerged as major focus in cancer research. However, the lack of a simple to use, sensitive and standardised assay to test for synthetic interactions has been slowing the efforts. Here we present a novel approach to synthetic lethality screening based on co-culturing two syngeneic cell lines containing individual fluorescent tags. By associating shRNAs for a target gene or control to individual fluorescence labels, we can easily follow individual cell fates upon siRNA treatment and high content imaging. We have demonstrated that the system can recapitulate the functional defects of the target gene depletion and is capable of discovering novel synthetic interactors and phenotypes. In a trial screen, we show that TIP60 exhibits synthetic lethality interaction with BAF180, and that in the absence of TIP60, there is an increase micronuclei dependent on the level of BAF180 loss, significantly above levels seen with BAF180 present. Moreover, the severity of the interactions correlates with proxy measurements of BAF180 knockdown efficacy, which may expand its usefulness to addressing synthetic interactions through titratable hypomorphic gene expression