Differences in autophagy marker levels at birth in preterm vs. term infants.

BACKGROUND: Preterm infants are susceptible to oxidative stress and prone to respiratory diseases. Autophagy is an important defense mechanism against oxidative-stress-induced cell damage and involved in lung development and respiratory morbidity. We hypothesized that autophagy marker levels differ between preterm and term infants. METHODS: In the prospective Basel-Bern Infant Lung Development (BILD) birth cohort we compared cord blood levels of macroautophagy (Beclin-1, LC3B), selective autophagy (p62) and regulation of autophagy (SIRT1) in 64 preterm and 453 term infants. RESULTS: Beclin-1 and LC3B did not differ between preterm and term infants. However, p62 was higher (0.37, 95% confidence interval (CI) 0.05;0.69 in log2-transformed level, p = 0.025, padj = 0.050) and SIRT1 lower in preterm infants (-0.55, 95% CI -0.78;-0.31 in log2-transformed level, padj < 0.001). Furthermore, p62 decreased (padj-value for smoothing function was 0.018) and SIRT1 increased (0.10, 95% CI 0.07;0.13 in log2-transformed level, padj < 0.001) with increasing gestational age. CONCLUSION: Our findings suggest differential levels of key autophagy markers between preterm and term infants. This adds to the knowledge of the sparsely studied field of autophagy mechanisms in preterm infants and might be linked to impaired oxidative stress response, preterm birth, impaired lung development and higher susceptibility to respiratory morbidity in preterm infants. IMPACT: To the best of our knowledge, this is the first study to investigate autophagy marker levels between human preterm and term infants in a large population-based sample in cord blood plasma This study demonstrates differential levels of key autophagy markers in preterm compared to term infants and an association with gestational age This may be linked to impaired oxidative stress response or developmental aspects and provide bases for future studies investigating the association with respiratory morbidity.

SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation.

During active DNA demethylation, 5-methylcytosine (5mC) is oxidized by TET proteins to 5-formyl-/5-carboxylcytosine (5fC/5caC) for replacement by unmethylated C by TDG-initiated DNA base excision repair (BER). Base excision generates fragile abasic sites (AP-sites) in DNA and has to be coordinated with subsequent repair steps to limit accumulation of genome destabilizing secondary DNA lesions. Here, we show that 5fC/5caC is generated at a high rate in genomes of differentiating mouse embryonic stem cells and that SUMOylation and the BER protein XRCC1 play critical roles in orchestrating TDG-initiated BER of these lesions. SUMOylation of XRCC1 facilitates physical interaction with TDG and promotes the assembly of a TDG-BER core complex. Within this TDG-BERosome, SUMO is transferred from XRCC1 and coupled to the SUMO acceptor lysine in TDG, promoting its dissociation while assuring the engagement of the BER machinery to complete demethylation. Although well-studied, the biological importance of TDG SUMOylation has remained obscure. Here, we demonstrate that SUMOylation of TDG suppresses DNA strand-break accumulation and toxicity to PARP inhibition in differentiating mESCs and is essential for neural lineage commitment.

Aberrant regulation of epigenetic modifiers contributes to the pathogenesis in patients with selenoprotein N-related myopathies.

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.

3CAPS - a structural AP-site analogue as a tool to investigate DNA base excision repair.

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3'-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase ß but repaired only by strand displacement as the 5'-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability.

Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to interact with transcription factors, histone acetyltransferases and de novo DNA methyltransferases, and it has been associated with DNA demethylation in gene promoters following activation of transcription, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.

Gadd45a promotes DNA demethylation through TDG.

Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)-initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

Extremely low-frequency magnetic fields and risk of childhood leukemia: A risk assessment by the ARIMMORA consortium.

Exposure to extremely low-frequency magnetic fields (ELF-MF) was evaluated in an International Agency for Research on Cancer (IARC) Monographs as "possibly carcinogenic to humans" in 2001, based on increased childhood leukemia risk observed in epidemiological studies. We conducted a hazard assessment using available scientific evidence published before March 2015, with inclusion of new research findings from the Advanced Research on Interaction Mechanisms of electroMagnetic exposures with Organisms for Risk Assessment (ARIMMORA) project. The IARC Monograph evaluation scheme was applied to hazard identification. In ARIMMORA for the first time, a transgenic mouse model was used to mimic the most common childhood leukemia: new pathogenic mechanisms were indicated, but more data are needed to draw definitive conclusions. Although experiments in different animal strains showed exposure-related decreases of CD8+ T-cells, a role in carcinogenesis must be further established. No direct damage of DNA by exposure was observed. Overall in the literature, there is limited evidence of carcinogenicity in humans and inadequate evidence of carcinogenicity in experimental animals, with only weak supporting evidence from mechanistic studies. New exposure data from ARIMMORA confirmed that if the association is nevertheless causal, up to 2% of childhood leukemias in Europe, as previously estimated, may be attributable to ELF-MF. In summary, ARIMMORA concludes that the relationship between ELF-MF and childhood leukemia remains consistent with possible carcinogenicity in humans. While this scientific uncertainty is dissatisfactory for science and public health, new mechanistic insight from ARIMMORA experiments points to future research that could provide a step-change in future assessments. Bioelectromagnetics. 37:183-189, 2016. © 2016 Wiley Periodicals, Inc.

Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability.

Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.

7,8-Dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases.

Hydroxyl radicals predominantly react with the C(8) of purines forming 7,8-dihydro-8-oxoguanine (8oxoG) and 7,8-dihydro-8-oxoadenine (8oxoA) adducts, which are highly mutagenic in mammalian cells. The majority of oxidized DNA bases are removed by DNA glycosylases in the base excision repair pathway. Here, we report for the first time that human thymine-DNA glycosylase (hTDG) and Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) can remove 8oxoA from 8oxoA•T, 8oxoA•G and 8oxoA•C pairs. Comparison of the kinetic parameters of the reaction indicates that full-length hTDG excises 8oxoA, 3,N(4)-ethenocytosine (εC) and T with similar efficiency (k(max) = 0.35, 0.36 and 0.16 min(-1), respectively) and is more proficient as compared with its bacterial homologue MUG. The N-terminal domain of the hTDG protein is essential for 8oxoA-DNA glycosylase activity, but not for εC repair. Interestingly, the TDG status had little or no effect on the proliferation rate of mouse embryonic fibroblasts after exposure to γ-irradiation. Nevertheless, using whole cell-free extracts from the DNA glycosylase-deficient murine embryonic fibroblasts and E. coli, we demonstrate that the excision of 8oxoA from 8oxoA•T and 8oxoA•G has an absolute requirement for TDG and MUG, respectively. The data establish that MUG and TDG can counteract the genotoxic effects of 8oxoA residues in vivo.

DNA-methylation variability in normal mucosa: a field cancerization marker in patients with adenomatous polyps.

BACKGROUND: The phenomenon of field cancerization reflects the transition of normal cells into those predisposed to cancer. Assessing the scope and intensity of this process in the colon may support risk prediction and colorectal cancer prevention. METHODS: The Swiss Epigenetic Colorectal Cancer Study (SWEPIC) study, encompassing 1111 participants for DNA methylation analysis and a subset of 84 for RNA sequencing, was employed to detect field cancerization in individuals with adenomatous polyps (AP). Methylation variations were evaluated for their discriminative capability, including in external cohorts, genomic localization, clinical correlations, and associated RNA expression patterns. RESULTS: Normal cecal tissue of individuals harboring an AP in the proximal colon manifested dysregulated DNA methylation compared to tissue from healthy individuals at 558 unique loci. Leveraging these adenoma-related differentially variable and methylated CpGs (aDVMCs), our classifier discerned between healthy and AP-adjacent tissues across SWEPIC datasets (cross-validated area under the receiver operating characteristic curve [ROC AUC] = 0.63-0.81), including within age-stratified cohorts. This discriminative capacity was validated in 3 external sets, differentiating healthy from cancer-adjacent tissue (ROC AUC = 0.82-0.88). Notably, aDVMC dysregulation correlated with polyp multiplicity. More than 50% of aDVMCs were significantly associated with age. These aDVMCs were enriched in active regions of the genome (P < .001), and associated genes exhibited altered expression in AP-adjacent tissues. CONCLUSIONS: Our findings underscore the early onset of field cancerization in the right colon during the neoplastic transformation process. A more extensive validation of aDVMC dysregulation as a stratification tool could pave the way for enhanced surveillance approaches, especially given its linkage to adenoma emergence.

Covalent PARylation of DNA base excision repair proteins regulates DNA demethylation.

The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation.

DNA glycosylases: in DNA repair and beyond.

The base excision repair machinery protects DNA in cells from the damaging effects of oxidation, alkylation, and deamination; it is specialized to fix single-base damage in the form of small chemical modifications. Base modifications can be mutagenic and/or cytotoxic, depending on how they interfere with the template function of the DNA during replication and transcription. DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy. All glycosylases share a common mode of action for damage recognition; they flip bases out of the DNA helix into a selective active site pocket, the architecture of which permits a sensitive detection of even minor base irregularities. Within the past few years, it has become clear that nature has exploited this ability to read the chemical structure of DNA bases for purposes other than canonical DNA repair. DNA glycosylases have been brought into context with molecular processes relating to innate and adaptive immunity as well as to the control of DNA methylation and epigenetic stability. Here, we summarize the key structural and mechanistic features of DNA glycosylases with a special focus on the mammalian enzymes, and then review the evidence for the newly emerging biological functions beyond the protection of genome integrity.

ELF exposure system for live cell imaging.

A programmable system has been developed for the study of both transient and persistent effects of extremely low frequency (ELF) magnetic field exposure of cell cultures. This high-precision exposure system enables experimental blinding and fully characterized exposure while simultaneously allowing live cell imaging. It is based on a live imaging cell around which two asymmetrical coils are wound in good thermal contact to a temperature-controlled water jacket, and is mounted on a microscope stage insert. The applied B-field uniformity of the active volume is better than 1.2% with an overall exposure uncertainty of less than 4.3% with very low transient field levels. The computer-controlled apparatus allows signal waveforms that are sinusoidal or composed of several harmonics, blind protocols, and monitoring of exposure and environmental conditions. B-fields up to 4 mT root mean square amplitude are possible with minimal temperature variation and no recognizable temperature differences between exposure and sham states. Sources of artifacts have been identified and quantified. There are no visible vibrations observable even at the highest magnifications and exposure levels.

Sperm chromatin accessibility's involvement in the intergenerational effects of stress hormone receptor activation.

Dexamethasone is a stress hormone receptor agonist used widely in clinics. We and others previously showed that paternal administration of dexamethasone in mice affects the phenotype of their offspring. The substrate of intergenerational transmission of environmentally induced effects often involves changes in sperm RNA, yet other epigenetic modifications in the germline can be affected and are also plausible candidates. First, we tested the involvement of altered sperm RNAs in the transmission of dexamethasone induced phenotypes across generations. We did this by injecting sperm RNA into naïve fertilized oocytes, before performing metabolic and behavioral phenotyping of the offspring. We observed phenotypic changes in discordance with those found in offspring generated by in vitro fertilization using sperm from dexamethasone exposed males. Second, we investigated the effect of dexamethasone on chromatin accessibility using ATAC sequencing and found significant changes at specific genomic features and gene regulatory loci. Employing q-RT-PCR, we show altered expression of a gene in the tissue of offspring affected by accessibility changes in sperm. Third, we establish a correlation between specific DNA modifications and stress hormone receptor activity as a likely contributing factor influencing sperm accessibility. Finally, we independently investigated this dependency by genetically reducing thymine-DNA glycosylase levels and observing concomitant changes at the level of chromatin accessibility and stress hormone receptor activity.

Hierarchical contribution of individual lifestyle factors and their interactions on adenomatous and serrated polyp risk.

BACKGROUND: Individual colorectal polyp risk factors are well characterized; however, insights into their pathway-specific interactions are scarce. We aimed to identify the impact of individual risk factors and their joint effects on adenomatous (AP) and serrated polyp (SP) risk. METHODS: We collected information on 363 lifestyle and metabolic parameters from 1597 colonoscopy participants, resulting in over 521,000 data points. We used multivariate statistics and machine-learning approaches to assess associations of single variables and their interactions with AP and SP risk. RESULTS: Individual factors and their interactions showed common and polyp subtype-specific effects. Abdominal obesity, high body mass index (BMI), metabolic syndrome, and red meat consumption globally increased polyp risk. Age, gender, and western diet associated with AP risk, while smoking was associated with SP risk. CRC family history was associated with advanced adenomas and diabetes with sessile serrated lesions. Regarding lifestyle factor interactions, no lifestyle or dietary adjustments mitigated the adverse smoking effect on SP risk, whereas its negative effect was exacerbated by alcohol in the conventional pathway. The adverse effect of red meat on SP risk was not ameliorated by any factor, but was further exacerbated by western diet along the conventional pathway. No modification of any factor reduced the negative impact of metabolic syndrome on AP risk, whereas increased fatless fish or meat substitutes' intake mitigated its effect on SP risk. CONCLUSIONS: Individual risk factors and their interactions for polyp formation along the adenomatous and serrated pathways are strongly heterogeneous. Our findings may facilitate tailored lifestyle recommendations and contribute to a better understanding of how risk factor combinations impact colorectal carcinogenesis.

Base excision by thymine DNA glycosylase mediates DNA-directed cytotoxicity of 5-fluorouracil.

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU-induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU-based chemotherapy.

DNA repair and the control of DNA methylation.

The successful establishment and stable maintenance of cell identity are critical for organismal development and tissue homeostasis. Cell identity is provided by epigenetic mechanisms that facilitate a selective readout of the genome. Operating at the level of chromatin, they establish defined gene expression programs during cell differentiation. Among the epigenetic modifications in mammalian chromatin, the 5'-methylation of cytosine in CpG dinucleotides is unique in that it affects the DNA rather than histones and the biochemistry of the DNA methylating enzymes offers a mechanistic explanation for stable inheritance. Yet, DNA methylation states appear to be more dynamic and their maintenance more complex than existing models predict. Also, methylation patterns are by far not always faithfully inherited, as best exemplified by human cancers. Often, these show widespread hypo- or hypermethylation across their genomes, reflecting an underlying epigenetic instability that may have contributed to carcinogenesis. The phenotype of unstable methylation in cancer illustrates the importance of quality control in the DNA methylation system and implies the existence of proof-reading mechanisms that enforce fidelity to DNA methylation in healthy tissue. Fidelity seems particularly important in islands of unmethylated CpG-rich sequences where an accurate maintenance of un- or differentially methylated states is critical for stable expression of nearby genes. Methylation proof-reading in such sequences requires a system capable of recognition and active demethylation of erroneously methylated CpGs. Active demethylation of 5-methylcytosine has been known to occur for long, but the underlying mechanisms have remained enigmatic and controversial. However, recent progress in this direction substantiates a role of DNA repair in such processes. This review will address general aspects of cytosine methylation stability in mammalian DNA and explore a putative role of DNA repair in methylation control.

Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function.

Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.

DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure.

Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage.