Note: Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin

Note: Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin

(doi: 10.1093/nar/gkn550)

Three PI3KK can phosphorylate H2Ax; ATM, ATR and DNA-PK – each requires partner to recognize the break differently. MRN-ATM (heterochromatin region, S/G2-phase), ATRIP-ATR (stalled replication fork or bulk-damage entering to S-phase) and KU70/80-DNA-PK (occur throughout cell cycle).

Scoring gH2Ax requires attention since it is not solely represented DSB. Remaining of gH2Ax foci does not mean the DSB has not been fixed. It may remain there to signal the HR to do more additional repair after seal with NHEJ, or the phosphatase system might be malfunction.

The further condensation of the 30nm fiber as well as higher levels of chromatin condensation, which culminate with the 10 000-fold compaction of the stretched DNA fiber in the 700nm metaphase chromosomes, are less well understood, but are facilitated by the linker histone H1 and condensins.

By organizing DNA, histones and nonhistone proteins generate a structural barrier to thousands of DNA-binding factors and DNA enzymes, whose uncontrolled access would compromise any meaningful activity and function of the DNA molecule.

organization of DNA into chromatin is instrumental for the multitude of gene expression patterns observed in the different cell lineages of multicellular organisms.

To ensure accessibility and chromatin structure facilitating the different functions of the DNA, interactions with core histones are regulated by numerous covalent modifications, many of which are localized at the amino terminal histone tails protruding from the nucleosome core, as well as by specialized variant core histones. These modifications reduce the affinity of histone tails for adjacent nucleosomes, thereby affecting chromatin structure.

Modification of chromatin structure has been extensively studied in the context of transcriptional regulation.

modification of chromatin structure also plays a central role in the regulation of DNA repair.

DNA lesions can be classified in two categories on the basis of their effect on chromatin integrity.

1.base and nucleotide damages as well as single interruptions of the sugar phosphate backbone, does not overly risk chromatin integrity or function, and error-free repair can be accommodated with limited, local modification of the chromatin structure using the complementary DNA strand as a template.

2.DNA double strand breaks (DSBs), but may also include some types of DNA-protein crosslinks, can bring chromatin to a state severely undermining its integrity and function.

Eukaryotic cells react to DNA damage with the so called DNA damage response (DDR), a sophisticated molecular circuitry developed to detect, signal and repair DNA damage.

DNA damage recognition and processing in the context of chromatin will require chromatin modification and will elicit events aiming at the coordination of checkpoint signaling with DNA repair or apoptosis.

The ultimate goal is the preservation of genomic integrity through the coupling of repair to other essential cellular metabolic activities such as gene expression, DNA replication, cell cycle progression and life or death decisions.

one that has been at the center of research activities during the last several years: the modification of

the H2A variant, H2AX. H2AX is one of the most conserved H2A-variants, and is present in chromatin at levels that vary between 2 and 25% of the H2A pool, depending on the cell line and tissue examined.

H2AX phosphorylation and g-H2AX foci formation are now generally accepted as consistent and quantitative markers of DSBs, applicable even under conditions where only a few DSBs are present.

the phosphorylation of H2AX occurs in the SQ motif that occupies a position of four residues from the carboxy terminus of the protein. SQ is followed by an acidic residue and the carboxy-terminus is hydrophobic.

It may be relevant that by virtue of the localization and orientation of H2A in the nucleosome, the S-139 phosphorylation will modify the nucleosome at a position right at the entry/exit points of the DNA.

Phosphorylated H2AX is detected after only a few minutes in cells exposed to IR, and phosphorylation reaches a maximum 30 min later.

One particularly relevant aspect of H2AX phosphorylation is that it is not limited to the immediate vicinity, but spreads instead to a large chromatin region surrounding the DSB.

that the modification spreads to a 2Mbp region of chromatin and comprises 2000 g-H2AX molecules

immunofluorescence analyses indicate that regions fifteen times larger (up to 30 Mbp) can be modified, implying that not every contiguous H2AX molecule is phosphorylated.

H2AX is not distributed randomly throughout bulk chromatin but exists in distinct clusters that define the boundaries of g-H2AX spreading.

In principle, all three major PIKK members, ATM, ATR and DNA-PKcs, have the potential of phosphorylating H2AX, and there is evidence that each of them actually carries out this phosphorylation when others are genetically compromised.

ATM seems best suited for H2AX phosphorylation by virtue of its ability to become activated by immediate, local chromatin modifications associated with DNA breakage.

DNAPKcs on the other hand, which become activated through its interaction with Ku, after Ku has directly bound to the DNA ends, is likely to have a reduced phosphorylation range requiring longer times for H2AX phosphorylation at longer distances.

ATM seems to be the main kinase associated with H2AX phosphorylation under normal physiological conditions. Furthermore, formation of g-H2AX triggered by uncapped telomeres, as well as meiotic recombination-associated DSBs are largely dependent on ATM.

DSBs associated with replication stress or UV damage are likely to be detected by ATR, which upon activation also phosphorylates H2AX.

ATR is activated through interaction with ATRIP, which recognizes single-stranded regions in the DNA. Such single-stranded regions can arise at stalled replication forks and also following repair of bulky DNA lesions. As such H2AX phosphorylation mediated by ATR does not necessarily reflect the presence of a

DSB in the genome. This is important to keep in mind when g-H2AX is scored as an indicator of DSBs, particularly in S-phase cells, and may partly explain the high number of g-H2AX normally seen in S-phase cells in the absence of DNA-damage-inducing treatments.

If phosphorylation of H2AX signals chromatin destabilization through a DSB, it should be reverted to H2A after repair restores chromatin integrity and structure. In mammalian cells, phosphatase 2A (PP2A) appears to be involved in the dephosphorylation of g-H2AX.

The exchange of H2AX with H2A has recently been shown to be mediated by FACT (for ‘FAcilitates Chromatin Transcription’), a heterodimer of Spt16 and SSRP1, and to be regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16.

Recent results also point to intriguing, highly specific molecular interactions that place g-H2AX at the early stages of the signaling response.

Although g-H2AX was shown to attract a number of proteins including NuA4, Ino80 and Swr1 of budding yeast, and the Tip60 chromatin remodeling complex of Drosophila, NBS1, 53BP1 and MDC1, the specificity of several of these interactions remains uncertain.

The forkhead-associated (FHA) domain recognizes phosphorylated threonine residues in a specific sequence context. In addition, it has been observed that two consecutive BRCT domains (BRCA1 C-terminal domain) can create a structural element with phosphor–peptide binding capacity.

Crystallography data nicely reveal how this tandem BRCT domain is precisely tailored to recognize the g-H2AX motif and demonstrate why the proximity of the phosphoserine to the C-terminus of H2AX remains invariable.

Although DNA-PK efficiently phosphorylates H2AX in the absence of ATM, it has been reported that it fails to do so when ATM is inhibited with the help of a specific inhibitor

Since free ends are likely to be initially recruited preferentially by Ku, some mechanism must exist to either block the interaction between DNA ends and Ku, or to facilitate their transition from Ku to the MRN complex. It is also notable that ATM and DNA-PKcs can be recruited to sites of DSBs by direct binding to MDC1 providing thus an additional level of interaction and regulation.

In addition to MDC1, 53BP1 also has the ability to detect changes in chromatin upon the induction of DSBs.

it is possible that g-H2AX-binding by MDC1 triggers changes in chromatin structure that lead to the exposure of the interaction-epitope for 53BP1.

While conjugation of ubiquitin chains via K48 generates a degradation signal, K63-linked polyubiquitin chains seem to be involved in DDR signaling.

The analysis of H2AX-deficient embryonic stem (ES) cells in mice showed that although H2AX is not essential for NHEJ or HRR, it does somehow modulate the efficiency of these repair pathways.

As a result, mouse cells lacking H2AX are radiosensitive and display deficits in DNA damage repair. Additionally, H2AX knock out mice show male-specific infertility and reduced levels of secondary immunoglobulin isotypes suggesting defects in class switch recombination (CSR).

efficient resolution of DSBs induced during CSR in lymphocytes requires H2AX, and its absence is associated with chromosome abnormalities involving the immunoglobulin locus.

the fact that direct effects of H2AX deficiency on DNA repair are subtle suggests that g-H2AX supports repair of selected DSBs and/or that it specifically assists specific repair pathways.

scoring of g-H2AX foci comes with some distinctive advantages, but it is also associated with short comings that should be carefully considered in highly quantitative or purely mechanistic studies.

Physical methods of DSB quantification typically have sensitivities that require the use of doses above 5Gy for a reliable assessment of the rejoining kinetics.

In addition, physical methods of DSB detection require DNA free of histones and other DNA associated proteins which is usually achieved by lysis at high temperatures. It has been suggested that lysis at high temperatures transforms to DSBs heat labile lesions, which can confound the assessment of the rejoining kinetics.

Despite these advantages of g-H2AX as a marker of DSBs, the method has limitations mainly because it does not follow the actual fate of the physical DSB, but rather registers cellular metabolic activities initiated to facilitate and optimize DSB repair.

the delayed kinetics of g-H2AX foci development can be rationally explained by the time required to initiate and to sustain the precisely coordinated biochemical events leading to the development of a mature focus.

Discrepancies between g-H2AX foci decay and DSB removal have also been reported in some cell systems

in the absence of treatment.

It can be hypothesized that g-H2AX continues marking the sites of some DSBs after resealing by NHEJ to facilitate additional processing by HRR.

An additional confounding factor in the analysis of induction and repair of DSBs via g-H2AX foci quantification comes from the observation that H2AX phosphorylation is diminished in areas of heterochromatin.

Weak H2AX phosphorylation in heterochromatin is also found in yeast, and in mouse fibroblasts, an increase of g-H2AX foci size is observed after chromatin becomes more accessible.

One of them is analysis and prediction of cell radiosensitivity to killing.

g-H2AX was unable to predict the efficacy of antioxidant radioprotective compounds. Thus, g-H2AX has the potential of developing to a useful predictor of cellular radiosensitivity to killing and may find application in the clinic during treatment of human tumors with ionizing radiation, in the evaluation of interindividual variations in radiosensitivity, in the analysis of the endogenous DSB load and even in the prediction of dose in nuclear accidents or terrorist attacks involving radioactive materials.