Chromatin remodeling finds its place in the DNA double-strand break response

The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans

Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl

<p>Abstract</p> <p>Background</p> <p>The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown.</p> <p>Results</p> <p>We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (<b>K</b>XXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase.</p> <p>Conclusion</p> <p>Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.</p

Human heterochromatin protein 1 isoforms HP1(Hsα) and HP1(Hsβ) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation

Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsα) or HP1(Hsβ), but not HP1(Hsγ), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsα) or HP1(Hsβ) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-ΔHP1(Hsα), GFP-ΔHP1(Hsβ), or GFP-ΔHP1(Hsγ) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsα) or HP1(Hsβ). In contrast, cells expressing mutant GFP-ΔHP1(Hsα) or GFP-ΔHP1(Hsβ) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsα) or HP1(Hsβ) suppressed tumorigenicity, whereas expression of mutant HP1(Hsα) or HP1(Hsβ) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsα) or HP1(Hsβ) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state

Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes

Ataxia-telangiectasia mutated (ATM)–deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor α/δ locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient αβ T cells with translocations involving the T cell receptor α/δ locus is directly related to the number of T cell receptor α rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment

Phosphorylation of ribosomal protein S6 confers PARP inhibitor resistance in BRCA1-deficient cancers

Inhibition of poly(ADP-ribose) polymerase (PARP) is a promising therapeutic strategy for BRCA1 deficient cancers, however, the development of drug resistance limits clinical efficacy. Previously we found that the BRCA1-AKT1 pathway contributes to tumorigenesis and that the AKT1/mTOR is a novel therapeutic target for BRCA1-deficient cancers. Here, we report that phosphorylation of ribosomal protein S6, a mTOR downstream effector, is greatly increased in BRCA1 deficient cells resistant to PARP inhibition. Phosphorylation of S6 is associated with DNA damage and repair signaling during PARP inhibitor treatment. In BRCA1 deficient cells, expression of S6 lacking all five phosphorylatable sites renders the cells sensitive to PARP inhibitor and increases DNA damage signals. In addition, the S6 mutations reduce tumor formation induced by Brca1-deficiency in mice. Inhibition of S6 phosphorylation by rapamycin restores PARP sensitivity to resistant cells. Combined treatment with rapamycin and PARP inhibitor effectively suppresses BRCA1-deficient tumor growth in mice. These results provide evidence for a novel mechanism by which BRCA1 deficient cancers acquire drug resistance and suggest a new therapeutic strategy to circumvent resistance