Multiple source genes of HAmo SINE actively expanded and ongoing retroposition in cyprinid genomes relying on its partner LINE

Background  

We recently characterized HAmo SINE and its partner LINE in silver carp and bighead carp based on hybridization capture of repetitive elements from digested genomic DNA in solution using a bead-probe [1]. To reveal the distribution and evolutionary history of SINEs and LINEs in cyprinid genomes, we performed a multi-species search for HAmo SINE and its partner LINE using the bead-probe capture and internal-primer-SINE polymerase chain reaction (PCR) techniques.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells

The aim of this study was to identify in human cells common targets of histone H3 lysine 9 (H3-Lys9) methylation, a modification that is generally associated with gene silencing. After chromatin immunoprecipitation using an H3-Lys9 methylated antibody, we cloned the recovered DNA and sequenced 47 independent clones. Of these, 38 clones (81%) contained repetitive elements, either short interspersed transposable element (SINE or Alu elements), long terminal repeat (LTR), long interspersed transposable element (LINE), or satellite region (ALR/Alpha) DNA, and three additional clones were near Alu elements. Further characterization of these repetitive elements revealed that 32 clones (68%) were Alu repeats, corresponding to both old Alu (23 clones) and young Alu (9 clones) subfamilies. Association of H3-Lys9 methylation was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. In addition, we randomly selected 5 Alu repeats from the recovered clones and confirmed association with H3-Lys9 by PCR using primer sets flanking the Alu elements. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly decreased the level of H3-Lys9 methylation in the Alu elements, suggesting that H3-Lys9 methylation may be related to the suppression of Alu elements through DNA methylation. Thus H3-Lys9 methylation is enriched at human repetitive elements, particularly Alu elements, and may play a role in the suppression of recombination by these elements.     

SINE retroposons can be used in vivo as nucleation centers for de novo methylation

SINEs (short interspersed elements) are an abundant class of transposable elements found in a wide variety of eukaryotes. Using the genomic sequencing technique, we observed that plant S1 SINE retroposons mainly integrate in hypomethylated DNA regions and are targeted by methylases. Methylation can then spread from the SINE into flanking genomic sequences, creating distal epigenetic modifications. This methylation spreading is vectorially directed upstream or downstream of the S1 element, suggesting that it could be facilitated when a potentially good methylatable sequence is single stranded during DNA replication, particularly when located on the lagging strand. Replication of a short methylated DNA region could thus lead to the de novo methylation of upstream or downstream adjacent sequences.     

SINEs and LINEs cluster in distinct DNA fragments of Giemsa band size

By in situ hybridization, short interspersed repeated DNA elements (SINEs), exemplified by Alu repeats, are located principally in Giemsa-light human metaphase chromosome bands. In contrast, the L1 family of long interspersed repeats (LINEs) preferentially cluster in Giemsa-dark bands. These SINE/LINE patterns also generally correspond to early and later replication band patterns. In order to provide a molecular link between structurally visible chromosome bands and a framework of interspersed repeats, we investigated patterns of SINE and LINE hybridization using pulse-field gel electrophoresis (PFGE). Interspersed SINEs and LINEs hybridize with high intensity to specific size fragments of 0.2–3 megabase pairs (Mb). Using appropriate restriction enzymes and pulse-field conditions, a number of fragments were delineated that were either SINE or LINE rich, and were mutually exclusive. Control studies with a human endogenous retroviral repeat that is related in sequence to the major LINE family, delineated a subset of fragments of 0.07–0.4 Mb with unequal intensity. Thus these less numerous repeats also appear to cluster selectively in DNA domains that are larger than a chromosome loop (60–120 kb). In summary, PFGE studies independently confirm the clustering of interspersed repeats on contiguous DNA loops. Selective clustering of repeat motifs may contribute to special structural or functional properties of large chromosome domains, such as chromatin extension/condensation or replication characteristics. In some cases the DNA fragments defined by these repeats approach the size of tandem satellite arrays.     

Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome

Trinucleotide (AGC)_n microsatellites are found as 3′ tails of the artiodactyl short interspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)_n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)_n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)_n microsatellites with 2–4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)_n microsatellites, Enrichment for (AGC), microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.     

Pig genome analysis: differential distribution of SINE and LINE sequences is less pronounced than in the human and mouse genomes

The distribution of SINE and LINE sequences in the pig genome was examined by fluorescence in situ hybridization (FISH), interspersed repeat PCR, and restriction analysis of high molecular weight DNA. FISH revealed a largely uniform hybridization to the euchromatic chromosome regions with both interspersed repeats, although a bias toward the G-bands was observed for the LINE probe. Southern blots of inter-SINE and inter-LINE PCR products showed strong hybridization to LINE and SINE probes, respectively. High molecular weight DNA derived from a pig |m~ hamster hybrid cell line was cut with a panel of G + C and A + T rich rare cutter restriction enzymes, then run on a pulsed field gel and Southern blotted. Sequential hybridization with SINE and LINE probes showed that SINE hybridization was to relatively low molecular weight fragments with the G + C rich enzymes, whereas the LINE probe gave hybridization to significantly larger fragments produced by these enzymes. DNA samples digested with A + T rich enzymes gave essentially similar patterns with SINE and LINE probes. We conclude that the pattern of differential distribution of SINEs and LINEs, which has been described in man and mouse, does exist in the pig but is much less pronounced. Received: 25 April 1995 / Accepted: 1 September 1995     

Association between length of gestation and cervical DNA methylation of PTGER2 and LINE 1-HS

Worldwide, more than 1 in 10 infants is born prior to 37 weeks gestation. Preterm birth can lead to increased mortality risk and poor life-long health and neurodevelopmental outcomes. Whether environmental risk factors affect preterm birth through epigenetic phenomena is largely unstudied. We sought to determine whether preterm risk factors, such as smoke exposure and education, were associated with cervical DNA methylation in the prostaglandin E receptor 2 gene (PTGER2) and a repetitive element, long interspersed nuclear element-1 Homo sapiens-specific (LINE 1-HS). Second, we aimed to determine whether mid-pregnancy DNA methylation of these regions in cervical samples could predict the length of gestation. We obtained a cervical swab between 16–19 weeks gestation from 80 women participating in a Mexico City birth cohort, used pyrosequencing to analyze DNA methylation of PTGER2 and LINE 1-HS, and examined associations with maternal covariates. We used accelerated failure time models to analyze associations of DNA methylation with the length of gestation. DNA methylation of both sequences was associated with Pap smear inflammation. LINE 1-HS methylation was associated with smoke exposure, BMI and parity. In adjusted models, gestations were 3.3 days longer (95%CI 0.6, 6.0) for each interquartile range of PTGER2 DNA methylation. Higher LINE 1-HS methylation was associated with shorter gestations (-3.3 days, 95%CI -6.5, -0.2). In conclusion, cervical DNA methylation was associated with risk factors for preterm birth and the length of gestation.     

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.     

Diversification,evolution and methylation of short interspersed nuclear element families in sugar beet and related Amaranthaceae species

Short interspersed nuclear elements (SINEs) are non‐autonomous non‐long terminal repeat retrotransposons which are widely distributed in eukaryotic organisms. While SINEs have been intensively studied in animals, only limited information is available about plant SINEs. We analysed 22 SINE families from seven genomes of the Amaranthaceae family and identified 34 806 SINEs, including 19 549 full‐length copies. With the focus on sugar beet (Beta vulgaris), we performed a comparative analysis of the diversity, genomic and chromosomal organization and the methylation of SINEs to provide a detailed insight into the evolution and age of Amaranthaceae SINEs. The lengths of consensus sequences of SINEs range from 113 nucleotides (nt) up to 224 nt. The SINEs show dispersed distribution on all chromosomes but were found with higher incidence in subterminal euchromatic chromosome regions. The methylation of SINEs is increased compared with their flanking regions, and the strongest effect is visible for cytosines in the CHH context, indicating an involvement of asymmetric methylation in the silencing of SINEs.     

Isolation and characterization of active LINE and SINEs from the eel

Long interspersed elements (LINEs) and short interspersed elements (SINEs) are retrotransposons. These elements can mobilize by the "copy-and-paste" mechanism, in which their own RNA is reverse-transcribed into complementary DNA (cDNA). LINEs and SINEs not only are components of eukaryotic genomes but also drivers of genomic evolution. Thus, studies of the amplification mechanism of LINEs and SINEs are important for understanding eukaryotic genome evolution. Here we report the characterization of one LINE family (UnaL2) and two SINE families (UnaSINE1 and UnaSINE2) from the eel (Anguilla japonica) genome. UnaL2 is approximately 3.6 kilobases (kb) and encodes only one open reading frame (ORF). UnaL2 belongs to the stringent type--thought to be a major group of LINEs--and can mobilize in HeLa cells. We also show that UnaL2 and the two UnaSINEs have similar 3' tails, and that both UnaSINE1 and UnaSINE2 can be mobilized by UnaL2 in HeLa cells. These elements are thus useful for delineating the amplification mechanism of stringent type LINEs as well as that of SINEs.     

PCR Detection of DNAs of Animal Origin in Feed by Primers Based on Sequences of Short and Long Interspersed Repetitive Elements

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.     

Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R(2) = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.     

Retropositional parasitism of SINEs on LINEs: identification of SINEs and LINEs in elasmobranchs.

Some previously unidentified short interspersed repetitive elements (SINEs) and long interspersed repetitive element (LINEs) were isolated from various higher elasmobranchs (sharks, skates, and rays) and characterized. These SINEs, members of the HE1 SINE family, were tRNA-derived and were widespread in higher elasmobranches. The 3'-tail region of this SINE family was strongly conserved among elasmobranchs. The LINEs, members of the HER1 LINE family, encoded an amino acid sequence similar to that encoded by the chicken CR1 LINE family, and they contained a strongly conserved 3'-tail region in the 3' untranslated region. This tail region of the HER1 LINE family was almost identical to that of the HE1 SINE family. Thus, the HE1 SINE family and the HER1 LINE family provide a clear example of a pair of SINEs and LINEs that share the same tail region. Conservation of the secondary structures of the tail regions, as well as of the nucleotide sequences, between the HE1 SINE family and HER1 LINE family during evolution suggests that SINEs utilize the enzymatic machinery for retroposition of LINEs through the recognition of higher-order structures of the conserved 3'-tail region. A discussion is presented of the parasitism of SINEs on LINEs during the evolution of these retroposons.     

PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.     

Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.