Georgios Markopoulos

Retrotransposon RNA expression and evidence for retrotransposition events in human oocytes

Although human diseases of retrotransposition-derived etiology have been documented, retrotransposon RNA expression and the occurrence of retrotransposition events in the human oocyte are not studied. We investigated the RNA expression of L1 and HERV-K10 retrotransposons in human oocytes by RT-PCR analysis with designed primers. Using denucleated germinal vesicles (GVs), we detected RT-PCR products of expressed L1, HERV-K10 and, unexpectedly, SINE-R, VNTR and Alu (SVA) retrotransposons. Their transcript specificities were identified as such following RNA-FISH and their origin by cloning and sequence alignment analyses. Assessing the expression level in comparison with somatic cells by densitometry analysis, we found that although in normal lymphocytes and transformed HeLa cells their profile was in an order of L1 > HERV-K10 > SVA, remarkably this was reversed in oocytes. To investigate whether de novo retrotransposition events occur and reverse transcriptases are expressed in the human oocyte, we introduced in GVs either a retrotransposition active human L1 or mouse reverse transcriptase deficient-VL30 retrotransposon tagged with an EGFP-based retrotransposition cassette. Interestingly, in both the cases, we observed EGFP-positive oocytes, associated with an abnormal morphology for L1 and granulation for VL30, and the retrotransposition events were confirmed by PCR. Our results: (i) show that L1, HERV-K10 and SVA retrotransposons are transcriptionally expressed and (ii) provide evidence, for the first time, for retrotransposition events occurring in the human oocyte. These findings suggest that both, network of retrotransposon transcripts and controlled retrotranspositions, might serve important functions required for oocyte development and fertilization while the uncontrolled ones might explain the onset of genetic disorders.

VL30 retrotransposition signals activation of a caspase-independent and p53-dependent death pathway associated with mitochondrial and lysosomal damage

The impact of long terminal repeat (LTR) retrotransposition on cell fate is unknown. Here, we investigated the effect of VL30 retrotransposition on cell death in SV40-transformed mouse SVTT1 cells. Transfection of a VL30 retrotransposon decreased the clonogenicity of SVTT1 by 17-fold, as compared to parental NIH3T3 cells. Correlated levels of retrotransposition frequency and cell death rates were found in retrotransposition-positive SVTT1 cloned cells, exhibiting DNA fragmentation, nuclear condensation, multinucleation and cytoplasmic vacuolization. Analysis of activation of effector caspases revealed a caspase-independent cell death mechanism. However, cell death was associated with p53 induction and concomitant upregulation of PUMAα and Bax and downregulation of Bcl-2 and Hsp70 protein expression. Moreover, we found partial loss of colocalization of large T-antigen (LT)/p53 and p53 translocation to mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP) accompanied by lysosomal membrane permeabilization (LMP). Interestingly, treatment with the antioxidant N-acetylcysteine abolished cell death, suggesting the involvement of mitochondrial-derived reactive oxygen species, and resulted in an increase of retrotransposition frequency. Importantly, the induction of cell death was VL30 retrotransposon-specific as VL30 mobilization was induced; in contrast, mobilization of the non-LTR L1 (LINE-1, long interspersed nuclear element-1), B2 and LTR MusD retrotransposons decreased. Our results provide, for the first time, strong evidence that VL30 retrotransposition mediates cell death via mitochondrial and lysosomal damage, uncovering the role of retrotransposition as a nuclear signal activating a mitochondrial-lysosomal crosstalk in triggering cell death.

Arsenic induces VL30 retrotransposition: the involvement of oxidative stress and heat-shock protein 70.

Arsenic is an environmental contaminant with known cytotoxic and carcinogenic properties, but the cellular mechanisms of its action are not fully known. As retrotransposition consists a potent mutagenic factor affecting genome stability, we investigated the effect of arsenic on retrotransposition of an enhanced green fluorescent protein (EGFP)-tagged nonautonomous long terminal repeat (LTR)-retrotransposon viral-like 30 (VL30) in a mouse NIH3T3 cell culture-retrotransposition assay. Flow cytometry analysis of assay cells treated with 2.5-20μM sodium arsenite revealed induction of retrotransposition events in a dose- and time-dependent manner, which was further confirmed as genomic integrations by PCR analysis and appearance of EGFP-positive cells by UV microscopy. Specifically, 20μM sodium arsenite strongly induced the VL30 retrotransposition frequency, which was ~90,000-fold higher than the natural one and also VL30 RNA expression was ~6.6-fold. Inhibition of the activity of endogenous reverse transcriptases by efavirenz at 15μM or nevirapine at 375μM suppressed the arsenite-induced VL30 retrotransposition by 71.16 or 79.88%, respectively. In addition, the antioxidant N-acetyl-cysteine reduced the level of arsenite-induced retrotransposition, which correlated with the rescue of arsenite-induced G2/M cell cycle arrest and cell toxicity. Treatment of assay cells ectopically overexpressing the human heat-shock protein 70 (Hsp70) with 15μM sodium arsenite resulted in an additional ~4.5-fold induction of retrotransposition compared with normal assay cells, whereas treatment with 20μM produced a massive cell death. Our results show for the first time that arsenic both as an oxidative and heat-shock mimicking agent is a potent inducer of VL30 retrotransposition in mouse cells. The impact of arsenic-induced retrotransposition, as a cellular response, on contribution to or explanation of the arsenic-associated toxicity and carcinogenicity is discussed.

Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts

Abstract Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL‐1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G1/S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL‐1 showed that a distinct set of 15 miRNAs were significantly up‐regulated in senescent cells including hsa‐let‐7d‐5p, hsa‐let‐7e‐5p, hsa‐miR‐23a‐3p, hsa‐miR‐34a‐5p, hsa‐miR‐122‐5p, hsa‐miR‐125a‐3p, hsa‐miR‐125a‐5p, hsa‐miR‐125b‐5p, hsa‐miR‐181a‐5p, hsa‐miR‐221‐3p, hsa‐miR‐222‐3p, hsa‐miR‐503‐5p, hsa‐miR‐574‐3p, hsa‐miR‐574‐5p and hsa‐miR‐4454. Importantly, pathway analysis of miRNA target genes down‐regulated during replicative senescence in a public RNA‐seq data set revealed a significant high number of genes regulating cell cycle progression, both G1/S and G2/M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative‐stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down‐regulation of cell cycle effectors correlated with the up‐regulation of miR‐221 upon both replicative and oxidative stress‐induced senescence. Transient expression of miR‐221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up‐regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype. HighlightsIdentification of a small set of senescence‐associated microRNAs (SA‐miRs).SA‐miRs target genes regulating cell cycle progression and telomere maintenance.MiR‐221 is up‐regulated during replicative and oxidative stress induced senescenceMiR‐221/222 targets cell cycle effectors.

Abnormal DLK1/MEG3 imprinting correlates with decreased HERV-K methylation after assisted reproduction and preimplantation genetic diagnosis

Abstract Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an important role in retrotransposon silencing and genomic imprinting during mammalian development. Assisted reproduction technologies (ARTs) may be associated with increased stress and risk of epigenetic changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and imprinted genes. Here, we investigated whether the methylation status of Human Endogenous Retroviruses (HERV)-K LTR retrotransposons and the imprinting signatures of the DLK1/MEG3. p57KIP2 and IGF2/H19 gene loci are linked during early human embryogenesis by examining trophoblast samples from ART pregnancies and preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status. Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to controls and this was associated with altered HERV-K methylation. No differences were detected in p57KIP2 and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using bioinformatics, we found that while the genome surrounding the p57KIP2 and IGF2/H19 genes differentially methylated regions had low coverage in transposable element (TE) sequences, the respective one of DLK1/MEG3 was characterized by an almost 2-fold higher coverage. Moreover, our analyses revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that TEs may be affecting the establishment of genomic imprinting under stress conditions.

A novel splicing isoform of protein arginine methyltransferase 1 (PRMT1) that lacks the dimerization arm and correlates with cellular malignancy

Methylation of arginine residues is an important modulator of protein function that is involved in epigenetic gene regulation, DNA damage response and RNA maturation, as well as in cellular signaling. The enzymes that catalyze this post‐translational modification are called protein arginine methyltransferases (PRMTs), of which PRMT1 is the predominant enzyme. Human PRMT1 has previously been shown to occur in seven splicing isoforms, which are differentially abundant in different tissues, and have distinct substrate specificity and intracellular localization. Here we characterize a novel splicing isoform which does not affect the amino‐terminus of the protein like the seven known isoforms, but rather lacks exons 8 and 9 which encode the dimerization arm of the enzyme that is essential for enzymatic activity. Consequently, the isoform does not form catalytically active oligomers with the other endogenous PRMT1 isoforms. Photobleaching experiments reveal an immobile fraction of the enzyme in the nucleus, in accordance with earlier results from our laboratory that had shown a tight association of inhibited or inactivated PRMT1 with chromatin and the nuclear scaffold. Thus, it apparently is able to bind to the same substrates as catalytically active PRMT1. This isoform is found in a variety of cell lines, but is increased in those of cancer origin or after expression of the EMT‐inducing transcriptional repressor Snail1. We discuss that the novel isoform could act as a modulator of PRMT1 activity in cancer cells by acting as a competitive inhibitor that shields substrates from access to active PRMT1 oligomers.

Roles of NF-κB Signaling in the Regulation of miRNAs Impacting on Inflammation in Cancer

The NF-κB family of transcription factors regulate the expression of genes encoding proteins and microRNAs (miRNA, miR) precursors that may either positively or negatively regulate a variety of biological processes such as cell cycle progression, cell survival, and cell differentiation. The NF-κB-miRNA transcriptional regulatory network has been implicated in the regulation of proinflammatory, immune, and stress-like responses. Gene regulation by miRNAs has emerged as an additional epigenetic mechanism at the post-transcriptional level. The expression of miRNAs can be regulated by specific transcription factors (TFs), including the NF-κB TF family, and vice versa. The interplay between TFs and miRNAs creates positive or negative feedback loops and also regulatory networks, which can control cell fate. In the current review, we discuss the impact of NF-κB-miRNA interplay and feedback loops and networks impacting on inflammation in cancer. We provide several paradigms of specific NF-κB-miRNA networks that can regulate inflammation linked to cancer. For example, the NF-κB-miR-146 and NF-κB-miR-155 networks fine-tune the activity, intensity, and duration of inflammation, while the NF-κB-miR-21 and NF-κB-miR-181b-1 amplifying loops link inflammation to cancer; and p53- or NF-κB-regulated miRNAs interconnect these pathways and may shift the balance to cancer development or tumor suppression. The availability of genomic data may be useful to verify and find novel interactions, and provide a catalogue of 162 miRNAs targeting and 40 miRNAs possibly regulated by NF-κB. We propose that studying active TF-miRNA transcriptional regulatory networks such as NF-κB-miRNA networks in specific cancer types can contribute to our further understanding of the regulatory interplay between inflammation and cancer, and also perhaps lead to the development of pharmacologically novel therapeutic approaches to combat cancer.

H2O2 signals via iron induction of VL30 retrotransposition correlated with cytotoxicity.

The impact of oxidative stress on mobilization of endogenous retroviruses and their effects on cell fate is unknown. We investigated the action of H2O2 on retrotransposition of an EGFP-tagged mouse LTR-retrotransposon, VL30, in an NIH3T3 cell-retrotransposition assay. H2O2 treatment of assay cells caused specific retrotranspositions documented by UV microscopy and PCR analysis. Flow cytometric analysis revealed an unusually high dose- and time-dependent retrotransposition frequency induced, ∼420,000-fold at 40 μM H2O2 compared to the natural frequency, which was reduced by ectopic expression of catalase. Remarkably, H2O2 moderately induced the RNA expression of retrotransposon B2 without affecting the basal expression of VL30s and L1 and significantly induced the expression of various endogenous reverse transcriptase genes. Further, whereas treatment with 50 μM FeCl2 alone was ineffective, cotreatment with 10 μM H2O2 and 50 μM FeCl2 caused a 6-fold higher retrotransposition induction than H2O2 alone, which was associated with cytotoxicity. H2O2- or H2O2/FeCl2-induced retrotransposition was significantly reduced by the iron chelator DFO or the antioxidant NAC, respectively. Furthermore, both H2O2-induced retrotransposition and associated cytotoxicity were inhibited after pretreatment of cells with DFO or the reverse transcriptase inhibitors efavirenz and etravirine. Our data show for the first time that H2O2, acting via iron, is a potent stimulus of retrotransposition contributing to oxidative stress-induced cell damage.

An Interstitial 4q31.21q31.22 Microdeletion Associated with Developmental Delay: Case Report and Literature Review

The 4q deletion syndrome phenotype consists of growth failure and developmental delay, minor craniofacial dysmorphism, digital anomalies, and cardiac and skeletal defects. We have identified an inversion (inv(1)(q25.2q31.1)) and an interstitial deletion in a boy with developmental delay using array-comparative genomic hybridization. This de novo deletion is located at 4q31.21q31.22 (145,963,820- 147,044,764), its size is 0.9-1.1 Mb, and it contains 7 genes (ABCE1, OTUD4, SMAD1, MMAA, C4orf51, ZNF827, and ANAPC10) as well as 5 retrotransposon-derived pseudogenes. Bioinformatic analysis revealed that while small copy number variations seem to have no impact on the phenotype, larger deletions or duplications in the deleted region are associated with developmental delay. Additionally, we found a higher coverage in transposable element sequences in the 4q31.21q31.22 region compared to that of the expected repeat density when regarding any random genome region. Transposable elements might have contributed to the reshaping of the genome architecture and, most importantly, we identified 3 L1PA family members in the breakpoint regions, suggesting their possible contribution in the mechanism underlying the appearance of this deletion. In conclusion, this is one of the smallest deletions reported associated with developmental delay, and we discuss the possible role of genomic features having an impact on the phenotype.

Impact of ZBTB7A hypomethylation and expression patterns on treatment response to hydroxyurea

BackgroundWe aimed to clarify the emerging epigenetic landscape in a group of genes classified as “modifier genes” of the β-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/β-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented.ResultsWe examined the CpG islands’ DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients’ monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU.ConclusionsThis is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5′ CpG sequences of all studied human HBB cluster “modifier genes.” Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the β-type hemoglobinopathy patients.