A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes.

Integration of transposable elements into the genome is mutagenic. Mechanisms targeting integrations into relatively safe locations, hence minimizing deleterious consequences for cell fitness, have emerged during evolution. In budding yeast, integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)-transcribed genes requires interaction between Ty1 integrase (IN1) and AC40, a subunit common to Pol I and Pol III. Here, we identify the Ty1 targeting domain of IN1 that ensures (i) IN1 binding to Pol I and Pol III through AC40, (ii) IN1 genome-wide recruitment to Pol I- and Pol III-transcribed genes, and (iii) Ty1 integration only at Pol III-transcribed genes, while IN1 recruitment by AC40 is insufficient to target Ty1 integration into Pol I-transcribed genes. Swapping the targeting domains between Ty5 and Ty1 integrases causes Ty5 integration at Pol III-transcribed genes, indicating that the targeting domain of IN1 alone confers Ty1 integration site specificity.

A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription.

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposons under tight transcriptional control.

Integration site selection by retroviruses and transposable elements in eukaryotes.

Transposable elements and retroviruses are found in most genomes, can be pathogenic and are widely used as gene-delivery and functional genomics tools. Exploring whether these genetic elements target specific genomic sites for integration and how this preference is achieved is crucial to our understanding of genome evolution, somatic genome plasticity in cancer and ageing, host-parasite interactions and genome engineering applications. High-throughput profiling of integration sites by next-generation sequencing, combined with large-scale genomic data mining and cellular or biochemical approaches, has revealed that the insertions are usually non-random. The DNA sequence, chromatin and nuclear context, and cellular proteins cooperate in guiding integration in eukaryotic genomes, leading to a remarkable diversity of insertion site distribution and evolutionary strategies.

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro.

Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.

Light and shadow on the mechanisms of integration site selection in yeast Ty retrotransposon families.

Transposable elements are ubiquitous in genomes. Their successful expansion depends in part on their sites of integration in their host genome. In Saccharomyces cerevisiae, evolution has selected various strategies to target the five Ty LTR-retrotransposon families into gene-poor regions in a genome, where coding sequences occupy 70% of the DNA. The integration of Ty1/Ty2/Ty4 and Ty3 occurs upstream and at the transcription start site of the genes transcribed by RNA polymerase III, respectively. Ty5 has completely different integration site preferences, targeting heterochromatin regions. Here, we review the history that led to the identification of the cellular tethering factors that play a major role in anchoring Ty retrotransposons to their preferred sites. We also question the involvement of additional factors in the fine-tuning of the integration site selection, with several studies converging towards an importance of the structure and organization of the chromatin.

The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag.

BACKGROUND: Nuclear localization of Gag is a property shared by many retroviruses and retrotransposons. The importance of this stage for retroviral replication is still unknown, but studies on the Rous Sarcoma virus indicate that Gag might select the viral RNA genome for packaging in the nucleus. In the case of Foamy viruses, genome encapsidation is mediated by Gag C-terminal domain (CTD), which harbors three clusters of glycine and arginine residues named GR boxes (GRI-III). In this study we investigated how PFV Gag subnuclear distribution might be regulated. RESULTS: We show that the isolated GRI and GRIII boxes act as nucleolar localization signals. In contrast, both the entire Gag CTD and the isolated GRII box, which contains the chromatin-binding motif, target the nucleolus exclusively upon mutation of the evolutionary conserved arginine residue at position 540 (R540), which is a key determinant of FV Gag chromatin tethering. We also provide evidence that Gag localizes in the nucleolus during FV replication and uncovered that the viral protein interacts with and is methylated by Protein Arginine Methyltransferase 1 (PRMT1) in a manner that depends on the R540 residue. Finally, we show that PRMT1 depletion by RNA interference induces the concentration of Gag C-terminus in nucleoli. CONCLUSION: Altogether, our findings suggest that methylation by PRMT1 might finely tune the subnuclear distribution of Gag depending on the stage of the FV replication cycle. The role of this step for viral replication remains an open question.

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration.

The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. We report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Binding to IN1 associates with allosteric changes in Pol III that may affect its transcriptional activity. The C-terminal domain of subunit C11, involved in RNA cleavage, inserts into the Pol III funnel pore, providing evidence for a two-metal mechanism during RNA cleavage. Additionally, ordering next to C11 of an N-terminal portion from subunit C53 may explain the connection between these subunits during termination and reinitiation. Deletion of the C53 N-terminal region leads to reduced chromatin association of Pol III and IN1, and a major fall in Ty1 integration events. Our data support a model in which IN1 binding induces a Pol III configuration that may favor its retention on chromatin, thereby improving the likelihood of Ty1 integration.

Ancrage chromatinien et intégration rétrovirale : implication des protéines IN et Gag.

Integration into the genome of the host cell is an obligatory step in the replication of retroelements. This feature accounts for the fact that these elements are both potential mutagens as well as vectors suitable for long-term gene therapy. Recently, many studies have reported that proviral insertion is not random but, rather, targets specific regions in the genome. Additionally, it has become clear that this process is highly regulated at the molecular level. Both viral proteins and cellular factors participate in the integration step, explaining why different retroelements have distinct integration profiles. This review describes recent advances about the integration of retroelements, focusing particularly on the mechanisms involved in the selectivity and specificity of integration and the chromatin-anchoring step, which precedes the insertion of the provirus.

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition.

BACKGROUND: Transposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN). RESULTS: Here, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. CONCLUSION: Our study shows that Ty1 IN is phosphorylated, as observed for retroviral INs and highlights an important role of CK2 in the regulation of Ty1 retrotransposition. In addition, the proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells.

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

Severe adenine starvation activates Ty1 transcription and retrotransposition in Saccharomyces cerevisiae.

Ty1 retrotransposons of the yeast Saccharomyces cerevisiae are activated by different kinds of stress. Here we show that Ty1 transcription is stimulated under severe adenine starvation conditions. The Bas1 transcriptional activator, responsible for the induction of genes of the de novo AMP biosynthesis pathway (ADE) in the absence of adenine, is not involved in this response. Activation occurs mainly on Ty1 elements, whose expression is normally repressed by chromatin and is suppressed in a hta1-htb1Delta mutant that alters chromatin structure. Activation is also abolished in a snf2Delta mutant. Several regions of the Ty1 promoter are necessary to achieve full activation, suggesting that full integrity of the promoter sequences might be important for activation. Together, these observations are consistent with a model in which the activation mechanism involves chromatin remodeling at Ty1 promoters. The consequence of Ty1 transcriptional activation in response to adenine starvation is an increase in Ty1 cDNA levels and a relief of Ty1 dormancy. The retrotransposition of four native Ty1 elements increases in proportion to their increase in transcription. Implications for the regulation of Ty1 mobility by changes in Ty1 mRNA levels are discussed.

Genome-Wide Mapping of Yeast Retrotransposon Integration Target Sites.

Transposable elements (TEs) are present in virtually all organisms. TE integration into genomes contributes to their structure and evolution, but can also be harmful in some cases. Deciphering where and how TE integration is targeted is fundamental to understand their intricate relationship with their host and explore the outcome of TE mobility on genome evolution and cell fitness. In general, TEs display integration site preference, which differs between elements. High-throughput mapping of de novo insertions by deep sequencing has recently allowed identifying genome-wide integration preferences of several TEs. These studies have provided invaluable clues to address the molecular determinants of integration site preference. Here, we provide a step-by-step methodology to generate massive de novo insertion events and prepare a library of genomic DNA for next-generation sequencing. We also describe a primary bioinformatic procedure to map these insertions in the genome. The whole procedure comes from our recent work on the integration of Ty1 in Saccharomyces cerevisiae, but could be easily adapted to the study of other TEs.

Recurrent acquisition of cytosine methyltransferases into eukaryotic retrotransposons.

Transposable elements are in a constant arms race with the silencing mechanisms of their host genomes. One silencing mechanism commonly used by many eukaryotes is dependent on cytosine methylation, a covalent modification of DNA deposited by C5 cytosine methyltransferases (DNMTs). Here, we report how two distantly related eukaryotic lineages, dinoflagellates and charophytes, have independently incorporated DNMTs into the coding regions of distinct retrotransposon classes. Concomitantly, we show that dinoflagellates of the genus Symbiodinium have evolved cytosine methylation patterns unlike any other eukaryote, with most of the genome methylated at CG dinucleotides. Finally, we demonstrate the ability of retrotransposon DNMTs to methylate CGs de novo, suggesting that retrotransposons could self-methylate retrotranscribed DNA. Together, this is an example of how retrotransposons incorporate host-derived genes involved in DNA methylation. In some cases, this event could have implications for the composition and regulation of the host epigenomic environment.

Author Correction: Recurrent acquisition of cytosine methyltransferases into eukaryotic retrotransposons.

The original version of this Article contained an error in the spelling of the author Hongfei Li, which was incorrectly given as Fei Hong. This has now been corrected in both the PDF and HTML versions of the Article.

Differential effects of chromatin and Gcn4 on the 50-fold range of expression among individual yeast Ty1 retrotransposons.

Approximately 30 copies of the Ty1 retrotransposon are present in the genome of Saccharomyces cerevisiae. Previous studies gave insights into the global regulation of Ty1 transcription but provided no information on the behavior of individual genomic elements. This work shows that the expression of 31 individual Ty1 elements in S288C varies over a 50-fold range. Their transcription is repressed by chromatin structures, which are antagonized by the Swi/Snf and SAGA chromatin-modifying complexes in highly expressed Ty1 elements. These elements carry five potential Gcn4 binding sites in their promoter regions that are mostly absent in weakly expressed Ty1 copies. Consistent with this observation, Gcn4 activates the transcription of highly expressed Ty1 elements only. One of the potential Gcn4 binding sites acts as an upstream activating sequence in vivo and interacts with Gcn4 in vitro. Since Gcn4 has been shown to interact with Swi/Snf and SAGA, we predict that Gcn4 activates Ty1 transcription by targeting these complexes to specific Ty1 promoters.

Binding to RNA regulates Set1 function.

The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.

International Congress on Transposable elements (ICTE 2016) in Saint Malo: mobile elements under the sun of Brittany.

The third international conference on Transposable Elements (ICTE) was held 16-19 April 2016 in Saint Malo, France. Organized by the French Transposition Community (Research group of the CNRS: "Mobile genetic elements: from mechanism to populations, an integrative approach") and the French Society of Genetics, the conference's goal was to bring together researchers who study transposition in diverse organisms, using multiple experimental approaches. The meeting gathered 180 participants from all around the world. Most of them contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into six scientific sessions: "Taming mobile DNA: self and non-self recognition"; "Trans-generational inheritance"; "Mobile DNA genome structure and organization, from molecular mechanisms to applications"; "Remembrance of (retro)transposon past: mobile DNA in genome evolution"; and finally "The yin and the yang of mobile DNA in human health".