IAP Elements Research Articles

DNA methylation is dispensable for suppression of the agouti viable yellow controlling element in murine embryonic stem cells.

The agouti viable (Avy) locus is considered a model to understand how retroelements function as controlling elements in mammals. Epigenetic factors, principally CpG methylation, are widely held to play a dominant regulatory role in controlling the locus' activity. The purpose of this study was to examine its behavior in ES cells and determine if this locus could be exploited for use in screen-based investigations. We have derived multiple Avy ES cell lines from the C57BL/6 strain and generated a cell line carrying a GFP-reporter gene (Avy/AGFP). Use of the DNA demethylating drug 5-azacitidine on various ES cell lines does not induce either agouti or GFP expression. Methylation analysis reveals that although most lines display normal methylation at IAP elements in general, the Avy IAP element is essentially unmethylated. In addition, we find that different repeat compartments are epigenetically unstable in a number of derived cell lines.

Quiescence-Induced LncRNAs Trigger H4K20 Trimethylation and Transcriptional Silencing

A complex network of regulatory pathways links transcription to cell growth and proliferation. Here we show that cellular quiescence alters chromatin structure by promoting trimethylation of histone H4 at lysine 20 (H4K20me3). In contrast to pericentric or telomeric regions, recruitment of the H4K20 methyltransferase Suv4-20h2 to rRNA genes and IAP elements requires neither trimethylation of H3K9 nor interaction with HP1 proteins but depends on long noncoding RNAs (lncRNAs) that interact with Suv4-20h2. Growth factor deprivation and terminal differentiation lead to upregulation of these lncRNAs, increase in H4K20me3, and chromatin compaction. The results uncover a lncRNA-mediated mechanism that guides Suv4-20h2 to specific genomic loci to establish a more compact chromatin structure in growth-arrested cells.

The Methylation of DNA Repeat Elements is Sex-Dependent and Temporally Different in Response to X Radiation in Radiosensitive and Radioresistant Mouse Strains

The effects of ionizing radiation on DNA methylation are of importance due to the role that DNA methylation plays in maintaining genome stability, and the presence of aberrant DNA methylation in many cancers. There is limited evidence that radiation-sensitivity may influence the modulation of DNA methylation by ionizing radiation, resulting in a loss of methylation. The BALB/c, CBA and C57Bl/6 strains are the most commonly utilized mouse strains in radiation research and are classified as radiation sensitive (BALB/c and CBA) or radiation resistant (C57Bl/6). We present here the first direct comparison of changes in repeat element DNA methylation (L1, B1 and Intracisternal A Particle; IAP) over time in these three mouse strains after high-dose radiation exposure. Using a high-resolution melt assay, methylation of the spleen repeat elements was investigated between 1 and 14 days after whole-body irradiation with 1 Gy X rays. Our study demonstrated that rather than a loss of methylation at the elements, all strains exhibited an early increase in L1 methylation one day after irradiation. In the most radiosensitive strain (BALB/c) the increase was also detected at 6 days postirradiation. The radioresistant C57Bl/6 strain exhibited a loss of L1 methylation at 14 days postirradiation. Less extensive changes to the B1 and IAP elements were detected at various time points, and pyrosequencing revealed that the responses of the strains were influenced by sex, with the male BALB/c and CBA mice exhibiting a greater response to the irradiation. The results of our study do not support the hypothesis that the most radiosensitive strains exhibit the greatest loss of repeat element DNA methylation after exposure to high-dose radiation. While the exact mechanism and biological outcome of the changes in DNA methylation observed here are still to be elucidated, this study provides the first evidence that radiation exposure elicits time-dependent changes in the methylation of repeat elements that are influenced by the genetic background, gender and the type of repeat element investigated. Furthermore, it suggest that any induced changes may not be persistent.

Lsh regulates LTR retrotransposon repression independently of Dnmt3b function.

BackgroundDNA methylation contributes to genomic integrity by suppressing repeat-associated transposition. In addition to the canonical DNA methyltransferases, several auxiliary chromatin factors are required to maintain DNA methylation at intergenic and satellite repeats. The interaction between Lsh, a chromatin helicase, and the de novo methyltransferase Dnmt3b facilitates deposition of DNA methylation at stem cell genes, which are hypomethylated in Lsh−/− embryos. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences.ResultsWe mapped genome-wide DNA methylation patterns in Lsh−/− and Dnmt3b−/− somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh−/− cells: LTR -retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed in Lsh−/− cells. LTR hypomethylation in Dnmt3b−/− cells is moderate, whereas IAP, LINE-1 and satellite elements are hypomethylated but silent. Repressed LINE-1 elements in Lsh−/− cells gain H3K4me3, but H3K9me3 levels are unaltered, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh−/− cells.ConclusionsOur study emphasizes that regulation of repetitive elements by Lsh and DNA methylation is selective and context dependent. Silencing of repeats in somatic cells appears not to be critically dependent on Dnmt3b function. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 to enforce selective repeat silencing.

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription.

The Mouse IAPE Endogenous Retrovirus Can Infect Cells through Any of the Five GPI-Anchored EphrinA Proteins

The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family –but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.

The mouse IAPE endogenous retrovirus can infect cells through any of the five GPI-anchored EphrinA proteins

The IAPE (Intracisternal A-type Particles elements with an Envelope) family of murine endogenous retroelements is present at more than 200 copies in the mouse genome. We had previously identified a single copy that proved to be fully functional, i.e. which can generate viral particles budding out of the cell and infectious on a series of cells, including human cells. We also showed that IAPE are the progenitors of the highly reiterated IAP elements. The latter are now strictly intracellular retrotransposons, due to the loss of the envelope gene and re-localisation of the associated particles in the course of evolution. In the present study we searched for the cellular receptor of the IAPE elements, by using a lentiviral human cDNA library and a pseudotype assay on transduced cells. We identified Ephrin A4, a GPI-anchored molecule involved in several developmental processes, as a receptor for the IAPE pseudotypes. We also found that the other 4 members of the Ephrin A family –but not those of the closely related Ephrin B family- were also able to mediate IAPE cell entry, thus significantly increasing the amount of possible cell types susceptible to IAPE infection. We show that these include mouse germline cells, as illustrated by immunohistochemistry experiments, consistent with IAPE genomic amplification by successive re-infection. We propose that the uncovered properties of the identified receptors played a role in the accumulation of IAPE elements in the mouse genome, and in the survival of a functional copy.

The pluripotency marker Rex-1/Zfp42 negatively regulates expression of endogenous retroviruses (ERV) and ERV-associated genes

Background Expression of the Yyl-related zinc finger protein Rex1 is restricted to primary spermatocytes, the preimplantation embryo and various types of pluripotent cells in culture. In line with its association to more pluripotent subpopulations, it is widely used as a marker for embryonic stem cells. Rex1 has been reported to regulate Tsix expression in ES cells, and imprinted genes during preimplantation development. Almost half of the mammalian genome is derived from retroelements, of which 25% are endogenous retroviruses (ERVs). During preimplantation development, these elements are silenced by epigenetic modifications, but the factors controlling these events are largely unknown. Materials and methods We have studied the role of Rex-1 in mouse ES cells and during mouse preimplantation development, using microinyection of either over-expressing plasmids or siRNAs. Gene expression was measured by (semi)-quantitative PCR. To identify additional genes regulated by Rex-1, we have carried out genome-wide chromatin association studies in mouse ES cells. Gene association was subsequently analyzed by chromatin-immunoprecipitation assays. Results In ESC with reduced expression of Rex-1 we did not observe changes in the expression of a selection of pluripotency and differentiation markers, in contrast to increased expression of endogenous retroviruses muERV-L and MusD. We probed for Rex-1 binding to these elements using ChIP assays in ES cells. Rex-1 strongly associates to MuERV-L and to a lower extent to IAP and MusD elements. Furthermore, association was not seen in Rex-1 depleted or RA-treated ES cells. Rex-1 also regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex-1 function in preimplantation embryos. Preliminary analysis of the genomic loci identified indicates that Rex-1 associates with a set of genes regulated by degenerated viarl LTRs, whose expression is highly regulated during preimplantation development, generally displaying peak expression levels in the blastocyst. Surprisingly these same genes are not necessarily highly expressed in mouse ES cells, although Rex-1 depletion does alter expression levels. Conclusions Our results suggest that Rex-1 regulates expression of ERV and ERV LTR-associated genes in mouse ES cells and during mouse preimplantation development. We hypothesize that Rex-1 has evolved as a regulator of endogenous retroviral transcription. We propose that the Yy1 family of transcription factors and Rex-1 in particular may have co-evolved with ERV. Such a model suggests that Yy1 family members may be instrumental to control the spread of silencing from repetitive elements to neighbouring genes, or to orchestrate ERVmediated control of cellular genes during preimplantation development and desease. We provide a potential explanation for the strong conservation of Yy1 family members, and a potential co-evolution mechanism that has allowed co-optation of ERV-derived cis elements for specific developmental processes.

Retrotransposon-Induced Heterochromatin Spreading in the Mouse Revealed by Insertional Polymorphisms

The “arms race” relationship between transposable elements (TEs) and their host has promoted a series of epigenetic silencing mechanisms directed against TEs. Retrotransposons, a class of TEs, are often located in repressed regions and are thought to induce heterochromatin formation and spreading. However, direct evidence for TE–induced local heterochromatin in mammals is surprisingly scarce. To examine this phenomenon, we chose two mouse embryonic stem (ES) cell lines that possess insertionally polymorphic retrotransposons (IAP, ETn/MusD, and LINE elements) at specific loci in one cell line but not the other. Employing ChIP-seq data for these cell lines, we show that IAP elements robustly induce H3K9me3 and H4K20me3 marks in flanking genomic DNA. In contrast, such heterochromatin is not induced by LINE copies and only by a minority of polymorphic ETn/MusD copies. DNA methylation is independent of the presence of IAP copies, since it is present in flanking regions of both full and empty sites. Finally, such spreading into genes appears to be rare, since the transcriptional start sites of very few genes are less than one Kb from an IAP. However, the B3galtl gene is subject to transcriptional silencing via IAP-induced heterochromatin. Hence, although rare, IAP-induced local heterochromatin spreading into nearby genes may influence expression and, in turn, host fitness.

MLL2 Is Required in Oocytes for Bulk Histone 3 Lysine 4 Trimethylation and Transcriptional Silencing

Author SummaryIt is well established that gametes and early mammalian embryos undergo extensive epigenetic changes, which are changes in phenotype or gene expression that do not entail changes in DNA sequence. However, the machinery responsible for epigenetic modification in these situations is poorly understood. In mice, we conditionally deleted the histone 3 lysine 4 (H3K4) methyltransferase Mll2, an enzyme that alters DNA structure and packaging, either in gametes or in somatic cells of the ovary and also produced a mouse hypomorph expressing low levels of MLL2. We show that MLL2 is required in oocytes during gametogenesis and is also needed as a maternally derived factor during early development. Oocytes deficient in Mll2 display decreased methylation of H3K4 (H3K4me3) and show abnormal maturation and gene expression, in particular of pro-apoptotic factors. In addition, we demonstrate that embryonic genome activation is compromised in the absence of Mll2. Together our results identify MLL2 as one of the key players in the epigenetic reprogramming required for female fertility in the mouse.

Endogenous retroviruses: from infectious elements to bona fide genes with a physiological role

Endogenous Retroviruses (ERVs) are present in all higher eukaryotes where they represent a large fraction of the genomes (8% in humans, divided into about 100 families). They are remnants of ancestral infections by bona fide retroviruses that have infected the germline and have thereafter been transmitted in a Mendelian manner. Conversely, they are a reservoir from which new infectious retroviruses can arise via recombination. The similarity between ERVs and present-day infectious retroviruses is remarkable, in terms of genome organization, encoded proteins, structures of the associated virus-like particles, and to some extent replicative cycles. Although most ERVs are defective due to genetic drift, the ''molecular biology'' of functional elements that could be identified in the human and murine genomes will be presented. Some ERVs have retained infectious properties (e.g. the HERV-K, GLN and IAPE elements), whereas others have evolved toward retroelements with a strictly intracellular life cycle (e.g. MusD and IAP elements) with high insertional mutagenesis activity. The molecular basis for these evolutionary transformations has been characterized, as well as the putative and/or still-present progenitors of these ''intracellularized'' elements. A step further to retrovirus ''capture'' by the host genomes will be exemplified with the so-called syncytin genes, which are retroviral envelope genes that have been ancestrally ''hijacked'' by several mammalian species - including primates and rodents- and have been conserved over >30 millions years of evolution in a functional state: syncytins have a placenta-specific expression and a cell-cell fusogenic activity most probably involved in placentation and syncytiotrophoblast formation. Data based on knockout mice will be presented that lend support to this subverted and essential role of ERV genes in mammalian physiology, and on the convergent impact of these independently acquired elements. Finally, the refined mechanisms that have evolved for the host control of ERVs and related elements will be described, with special emphasis on the restriction effects of the cellular APOBEC3 cytosine deaminase proteins, which inhibit amplification of both the infectious ERVs and the intracellularized retroelements, and whose effects can be clearly detected through the signatures imprinted on the numerous genomic copies of these elements.

Restriction by APOBEC3 proteins of endogenous retroviruses with an extracellular life cycle: ex vivo effects and in vivo"traces" on the murine IAPE and human HERV-K elements

BackgroundAPOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons.ResultsHere we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an ex vivo assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. In silico analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses "traces" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member.ConclusionThese results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites.

Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor α

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129 mice were fed a control or WY-14,643-containing (1000 ppm) diet for one week, five weeks or five months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at five months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparα-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of Pparα.

Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse.

Genome-wide epigenetic reprogramming by demethylation occurs in early mouse embryos and primordial germ cells. In early embryos many single-copy sequences become demethylated both by active and passive demethylation, whereas imprinted gene methylation remains unaffected. In primordial germ cells single-copy and imprinted sequences are demethylated, presumably by active demethylation. Here we investigated systematically by bisulphite sequencing the methylation profiles of IAP and Line1 repeated sequence families during preimplantation and primordial germ cell development. Whereas Line1 elements were substantially demethylated during both developmental periods, IAP elements were largely resistant to demethylation, particularly during preimplantation development. This may be desirable in order to prevent IAP retrotransposition, which could cause mutations. In turn, this can result in the transgenerational inheritance of epigenetic states of IAPs, which could lead to heritable epimutations of neighbouring genes through influencing their transcriptional states.

Penetrance and low concordance in monozygotic twins in disease: are they the results of alterations in somatic genomes?

Penetrance and low concordance in monozygotic twins in disease: are they the results of alterations in somatic genomes?