Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.

Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.

DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

Altered gene expression in human placentas after IVF/ICSI.

STUDY QUESTION: Is gene expression in placental tissue of IVF/ICSI patients altered when compared with a spontaneously conceived group, and are these alterations due to loss of imprinting (LOI) in the case of imprinted genes? SUMMARY ANSWER: An altered imprinted gene expression of H19 and Pleckstrin homology-like domain family A member 2 (PHLDA2), which was not due to LOI, was observed in human placentas after IVF/ICSI and several biological pathways were significantly overrepresented and mostly up-regulated. WHAT IS KNOWN ALREADY: Genomic imprinting plays an important role in placental biology and in placental adaptive responses triggered by external stimuli. Changes in placental development and function can have dramatic effects on the fetus and its ability to cope with the intrauterine environment. An increased frequency of placenta-related problems as well as an adverse perinatal outcome is seen in IVF/ICSI derived pregnancies, but the role of placental epigenetic deregulation is not clear yet. STUDY DESIGN AND PARTICIPANTS: In this prospective cohort study, a total of 115 IVF/ICSI and 138 control couples were included during pregnancy. After applying several exclusion criteria (i.e. preterm birth or stillbirth, no placental samples, pregnancy complications or birth defects), respectively, 81 and 105 placentas from IVF/ICSI and control pregnancies remained for analysis. Saliva samples were collected from both parents. METHODS: We quantitatively analysed the mRNA expression of several growth-related imprinted genes [H19, insulin-like growth factor 2 (IGF2), PHLDA2, cyclin-dependent kinase inhibitor 1C (CDKN1C), mesoderm-specific transcript homolog (MEST) isoform α and ß by quantitative PCR] after standardization against three housekeeping genes [Succinate dehydrogenase A (SDHA), YWHAZ and TATA-binding protein (TBP)]. A quantitative allele-specific expression analysis of the differentially expressed imprinted genes was performed to investigate LOI, independent of the mechanism of imprinting. Furthermore, a microarray analysis was carried out (n = 10 in each group) to investigate the expression of non-imprinted genes as well. MAIN RESULTS AND THE ROLE OF CHANCE: Both H19 and PHLDA2 showed a significant change, respectively, a 1.3-fold (P = 0.033) and 1.5-fold (P = 0.002) increase in mRNA expression in the IVF/ICSI versus control group. However, we found no indication that there is an increased frequency of LOI in IVF/ICSI placental samples. Genome-wide mRNA expression revealed 13 significantly overrepresented biological pathways involved in metabolism, immune response, transmembrane signalling and cell cycle control, which were mostly up-regulated in the IVF/ICSI placental samples. LIMITATIONS, REASONS FOR CAUTION: Only a subset of samples was found to be fully informative, which unavoidably led to lower sample numbers for our LOI analysis. Our study cannot distinguish whether the reported differences in the IVF/ICSI group are exclusively attributable to the IVF/ICSI technique itself or to the underlying subfertility of the patients. WIDER IMPLICATIONS OF THE FINDINGS: Whether these placental adaptations observed in pregnancies conceived by IVF/ICSI might be connected to an adverse perinatal outcome after IVF remains unknown. However, it is possible that these differences affect fetal development and long-term patterns of gene expression, as well as maternal gestational physiology. STUDY FUNDING/COMPETING INTERESTS: Partly funded by an unrestricted research grant by Organon BV (now MSD BV) and GROW School for Oncology and Developmental Biology without any role in study design, data collection and analysis or preparation of the manuscript. No conflict of interests to declare. TRIAL REGISTRATION NUMBER: Dutch Trial Registry (NTR) number 1298.

C5-DNA methyltransferase inhibitors: from screening to effects on zebrafish embryo development.

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.

Mechanistic insights on the inhibition of c5 DNA methyltransferases by zebularine.

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.

Specific hypomethylated CpGs at the IGF2 locus act as an epigenetic biomarker for familial adenomatous polyposis colorectal cancer.

AIMS: The identification of specific biomarkers for colorectal cancer is of primary importance for early diagnosis. The aim of this study was to evaluate if methylation changes at the IGF2/H19 locus could be predictive for individuals at high risk for developing sporadic or hereditary colorectal cancer. MATERIALS & METHODS: Quantitative methylation analysis using pyrosequencing was performed on three differentially methylated regions (DMRs): IGF2 DMR0 and DMR2 and the H19 DMR in DNA samples from sporadic colorectal cancer (n = 26), familial adenomatous polyposis (n = 35) and hereditary nonpolyposis colorectal cancer (n = 19) patients. RESULTS: We report in this article for the first time, that in sporadic colorectal cancer tumor DNA both the IGF2 DMR0 and DMR2 are hypomethylated, while the H19 DMR retains its monoallelic methylation pattern. In lymphocyte DNA, a striking hypomethylation of nine contiguous correlated CpGs was found in the IGF2 DMR2 but only in familial adenomatous polyposis patients. CONCLUSION: Methylation alterations at the IGF2 locus are more extensive than previously reported and DMR2 hypomethylation in lymphocyte DNA might be a specific epigenetic biomarker for familial adenomatous polyposis patients.