Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity

L1 is a ubiquitous interspersed repeated sequence in mammals that achieved its high copy number by autonomous retrotransposition. Individual L1 elements within a genome differ in sequence and retrotransposition activity. Retrotransposition requires two L1-encoded proteins, ORF1p and ORF2p. Chimeric elements were used to map a 15-fold difference in retrotransposition efficiency between two L1 variants from the mouse genome, TFC and TFspa, to a single amino acid substitution in ORF1p, D159H. The steady-state levels of L1 RNA and protein do not differ significantly between these two elements, yet new insertions are detected earlier and at higher frequency in TFC, indicating that it converts expressed L1 intermediates more effectively into new insertions. The two ORF1 proteins were purified and their nucleic acid binding and chaperone activities were examined in vitro. Although the RNA and DNA oligonucleotide binding affinities of these two ORF1 proteins were largely indistinguishable, D159 was significantly more effective as a nucleic acid chaperone than H159. These findings support a requirement for ORF1p nucleic acid chaperone activity at a late step during L1 retrotransposition, extend the region of ORF1p that is known to be critical for its functional interactions with nucleic acids, and enhance understanding of nucleic acid chaperone activity

Exploring the gonad transcriptome of two extreme male pigs with RNA-seq

Background: Although RNA-seq greatly advances our understanding of complex transcriptome landscapes, such as those found in mammals, complete RNA-seq studies in livestock and in particular in the pig are still lacking. Here, we used high-throughput RNA sequencing to gain insight into the characterization of the poly-A RNA fraction expressed in pig male gonads. An expression analysis comparing different mapping approaches and detection of allele specific expression is also discussed in this study. Results: By sequencing testicle mRNA of two phenotypically extreme pigs, one Iberian and one Large White, we identified hundreds of unannotated protein-coding genes (PcGs) in intergenic regions, some of them presenting orthology with closely related species. Interestingly, we also detected 2047 putative long non-coding RNA (lncRNA), including 469 with human homologues. Two methods, DEGseq and Cufflinks, were used for analyzing expression. DEGseq identified 15% less expressed genes than Cufflinks, because DEGseq utilizes only unambiguously mapped reads. Moreover, a large fraction of the transcriptome is made up of transposable elements (14500 elements encountered), as has been reported in previous studies. Gene expression results between microarray and RNA-seq technologies were relatively well correlated (r = 0.71 across individuals). Differentially expressed genes between Large White and Iberian showed a significant overrepresentation of gamete production and lipid metabolism gene ontology categories. Finally, allelic imbalance was detected in ~ 4% of heterozygous sites. Conclusions: RNA-seq is a powerful tool to gain insight into complex transcriptomes. In addition to uncovering many unnanotated genes, our study allowed us to determine that a considerable fraction is made up of long non-coding transcripts and transposable elements. Their biological roles remain to be determined in future studies. In terms of differences in expression between Large White and Iberian pigs, these were largest for genes involved in spermatogenesis and lipid metabolism, which is consistent with phenotypic extreme differences in prolificacy and fat deposition between these two breeds

ECAT11/L1td1 Is Enriched in ESCs and Rapidly Activated During iPSCGeneration, but It Is Dispensable for the Maintenance and Induction of Pluripotency

The principal factors that lead to proliferation and pluripotency in embryonic stem cells (ESCs) have been vigorously investigated. However, the global network of factors and their full signaling cascade is still unclear. In this study, we found that ECAT11 (L1td1) is one of the ESC-associated transcripts harboring a truncated fragment of ORF-1, a component of theL1 retrotransposable element. We generated an ECAT11 knock-in mouse by replacing its coding region with green fluorescent protein. In the early stage of development, the fluorescence was observed at the inner cell mass of blastocysts and epiblasts. Despite this specific expression, ECAT11-null mice grow normally and are fertile. In addition, ECAT11 was dispensable for both the proliferation and pluripotency of ESCs.We found rapid and robust activation of ECAT11 in fibroblasts after the forced expression of transcription factors that can give rise pluripotency in somatic cells.However, iPS cells could be established from ECAT11-null fibroblasts. Our data demonstrate thedispensability of ECAT11/L1td1 in pluripotency, despite its specific expression

Somatic expression of LINE-1 elements in human tissues

LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated β-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging

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

Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark

Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements

A Novel Method of Distance Measurement Based on Pulse Position Modulation and Synchronization of Chaotic Signals Using Ultrasonic Radar Systems

This paper deals with a novel method of transmission and receipt of a signal based on both the property of two chaotic systems generating the same chaotic signal when they are synchronized and the property of pulse position modulation (PPM) to be insensitive to the distortions of the transmission channel. The method is discussed in the context of ultrasonic radar systems, in which the transmitter and receiver, which consist of ultrasonic sensors, are near each other, and the received signal consists of the transmitted signal reflected by an obstacle. A reference sinusoidal signal is superimposed to a chaotic signal generated by a master chaotic system, and the whole signal is modulated according to the PPM method and transmitted by the sensor. The received signal is demodulated, and the demodulated signal forces a slave chaotic system to generate the chaotic signal embedded in it, which allows recovery of the sinusoidal signal by subtracting this chaotic signal from the demodulated echo. The difference of the phases of the reference sinusoidal signal and the recovered sinusoidal signal allows computation of the time of flight of the signal and, consequently, the distance of the radar system from the obstacle. The novel method is illustrated and tested by both simulation and experiments. The interference problem between the considered radar system and other radar systems (crosstalk) is also addressed, and a solution is proposed to avoid it

Defects detection and characterization by using cellular neural networks

In this document, a new method to detect and to characterize surface defects in mechanical parts is reported. Cellular neural networks are used as tools for the implementation of the stereoscopic vision analysis technique. Suitable applications in microscopic defect analysis (real-time processing), in various fields (e.g. aeronautics applications) are introduced, by means of the reported examples in order to validate the cellular neural networks (CNN) approach. © 2001 IEEE