DNA Damage Leaves its Mark on Chromatin

DNA organization into chromatin has a major influence on the cellular response to DNA damage. Recent studies in various systems ranging from yeast to human cells stress the importance of chromatin not simply as a barrier to DNA repair processes but also as an active contributor to the DNA damage response. Indeed, modulations of chromatin organization involving various degrees of rearrangements, such as histone modifications and even nucleosome displacement, can promote efficient repair and also participate in checkpoint signaling. Here, we survey recent progress in delineating how chromatin rearrangements provide crosstalk with the DNA damage response. In particular, we highlight new data on histone dynamics at damage sites and discuss their functional importance for the stable propagation of specific chromatin states.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.     

Impact of Chromatin Structures on DNA Processing for Genomic Analyses

Chromatin has an impact on recombination, repair, replication, and evolution of DNA. Here we report that chromatin structure also affects laboratory DNA manipulation in ways that distort the results of chromatin immunoprecipitation (ChIP) experiments. We initially discovered this effect at the Saccharomyces cerevisiae HMR locus, where we found that silenced chromatin was refractory to shearing, relative to euchromatin. Using input samples from ChIP-Seq studies, we detected a similar bias throughout the heterochromatic portions of the yeast genome. We also observed significant chromatin-related effects at telomeres, protein binding sites, and genes, reflected in the variation of input-Seq coverage. Experimental tests of candidate regions showed that chromatin influenced shearing at some loci, and that chromatin could also lead to enriched or depleted DNA levels in prepared samples, independently of shearing effects. Our results suggested that assays relying on immunoprecipitation of chromatin will be biased by intrinsic differences between regions packaged into different chromatin structures - biases which have been largely ignored to date. These results established the pervasiveness of this bias genome-wide, and suggested that this bias can be used to detect differences in chromatin structures across the genome.     

Vitamin E Protects DNA from Oxidative Damage in Human Hepatocellular Carcinoma Cell Lines

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (?p<0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in α-tocopherol succinate (α-TS, 5?μM for 24?h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with α-TS, became resistant to ADP-Fe³⁺ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe³⁺ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5?cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with α-TS fully reverted this effect and significantly (p<0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500?cGy. Resistance to doxorubicin was not affected by α-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.     

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Objectives

to assess the cardioprotective properties of a blueberry enriched diet (BD).

Background

Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.

Methods and Results

Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (t_MPT) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed t_MPT. The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.

Conclusion

A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.     

Resveratrol Protects Mouse Oocytes from Methylglyoxal-Induced Oxidative Damage

Methylglyoxal, a reactive dicarbonyl compound, is mainly formed from glycolysis. Methylglyoxal can lead to the dysfunction of mitochondria, the depletion of cellular anti-oxidation enzymes and the formation of advanced glycation ends. Previous studies showed that the accumulation of methylglyoxal and advanced glycation ends can impair the oocyte maturation and reduce the oocyte quality in aged and diabetic females. In this study, we showed that resveratrol, a kind of phytoalexin found in the skin of grapes, red wine and other botanical extracts, can alleviate the adverse effects caused by methylglyoxal, such as inhibition of oocyte maturation and disruption of spindle assembly. Besides, methylglyoxal-treated oocytes displayed more DNA double strands breaks and this can also be decreased by treatment of resveratrol. Further investigation of these processes revealed that methylglyoxal may affect the oocyte quality by resulting in excessive reactive oxygen species production, aberrant mitochondrial distribution and high level lipid peroxidation, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can protect the oocytes from methylglyoxal-induced cytotoxicity and this was mainly through the correction of the abnormity of cellular reactive oxygen species metabolism.     

Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.     

NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.     

染色质免疫沉淀试验中基因组DNA超声破碎条件优化策略

染色质免疫沉淀试验中对共价交联的基因组染色质进行超声处理以获得适当大小的DNA片段是ChIPSeq和ChIP-chip成功的前提。影响基因组DNA破碎的因素很多,细胞浓度、细胞裂解方式、超声体积、探头深度、超声环境、超声时间和超声功率等都会对超声效果产生影响。如何获得各个可变参数的优化组合是一个需要多次摸索的过程,借鉴其它研究者已经建立的优化条件进行探索是一条比较省时简便的途径。本研究针对超声时间、细胞裂解方式、超声环境和超声功率等因素进行优化;利用优化的条件超声,得到适合大小的DNA片段;对这些片段进行染色质免疫沉淀试验,获得已知靶基因的特异性富集;证明超声优化条件是成功的。本研究提出的超声优化策略及获得的优化条件为后续ChIPSeq试验创造了前提条件,也对其他研究者具有借鉴意义。     

Investigating DNA Radiation Damage Using X-Ray Absorption Spectroscopy

The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.     

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence

1. Download high-res image (133KB)
2. Download full-size image

     

DNA Damage Responses following Exposure to Modulated Radiation Fields

During the delivery of advanced radiotherapy treatment techniques modulated beams are utilised to increase dose conformity across the target volume. Recent investigations have highlighted differential cellular responses to modulated radiation fields particularly in areas outside the primary treatment field that cannot be accounted for by scattered dose alone. In the present study, we determined the DNA damage response within the normal human fibroblast AG0-1522B and the prostate cancer cell line DU-145 utilising the DNA damage assay. Cells plated in slide flasks were exposed to 1 Gy uniform or modulated radiation fields. Modulated fields were delivered by shielding 25%, 50% or 75% of the flask during irradiation. The average number of 53BP1 or γH2AX foci was measured in 2 mm intervals across the slide area. Following 30 minutes after modulated radiation field exposure an increase in the average number of foci out-of-field was observed when compared to non-irradiated controls. In-field, a non-uniform response was observed with a significant decrease in the average number of foci compared to uniformly irradiated cells. Following 24 hrs after exposure there is evidence for two populations of responding cells to bystander signals in-and out-of-field. There was no significant difference in DNA damage response between 25%, 50% or 75% modulated fields. The response was dependent on cellular secreted intercellular signalling as physical inhibition of intercellular communication abrogated the observed response. Elevated residual DNA damage observed within out-of-field regions decreased following addition of an inducible nitric oxide synthase inhibitor (Aminoguanidine). These data show, for the first time, differential DNA damage responses in-and out-of-field following modulated radiation field delivery. This study provides further evidence for a role of intercellular communication in mediating cellular radiobiological response to modulated radiation fields and may inform the refinement of existing radiobiological models for the optimization of advanced radiotherapy treatment plans.