Telomere Maintenance in Organisms without Telomerase

Biochemistr

Chapter Telomere Maintenance in Organisms without Telomerase

Biochemistr

Non-long terminal repeat (non-LTR) retrotransposons: mechanisms, recent developments, and unanswered questions

Non-long terminal repeat (non-LTR) retrotransposons are present in most eukaryotic genomes. In some species, such as humans, these elements are the most abundant genome sequence and continue to replicate to this day, creating a source of endogenous mutations and potential genotoxic stress. This review will provide a general outline of the replicative cycle of non-LTR retrotransposons. Recent findings regarding the host regulation of non-LTR retrotransposons will be summarized. Finally, future directions of interest will be discussed

Silence at the End: How Drosophila Regulates Expression and Transposition of Telomeric Retroelements

The maintenance of chromosome ends in Drosophila is an exceptional phenomenon because it relies on the transposition of specialized retrotransposons rather than on the activity of the enzyme telomerase that maintains telomeres in almost every other eukaryotic species. Sequential transpositions of Het-A, TART, and TAHRE (HTT) onto chromosome ends produce long head-to-tail arrays that are reminiscent to the long arrays of short repeats produced by telomerase in other organisms. Coordinating the activation and silencing of the HTT array with the recruitment of telomere capping proteins favors proper telomere function. However, how this coordination is achieved is not well understood. Like other Drosophila retrotransposons, telomeric elements are regulated by the piRNA pathway. Remarkably, HTT arrays are both source of piRNA and targets of gene silencing thus making the regulation of Drosophila telomeric transposons a unique event among eukaryotes. Herein we will review the genetic and molecular mechanisms underlying the regulation of HTT transcription and transposition and will discuss the possibility of a crosstalk between piRNA mediated regulation, telomeric chromatin establishment and telomere protection

In vivo analysis of L1 retrotransposition in Huntington\u2019s disease mouse brain

Transposable elements (TEs) are mobile genetic elements that constitute a large fraction of eukaryotic genomes. TEs co-evolved with their host genomes, providing powerful tools of genome plasticity and regulation. Long Interspersed Nuclear Elements 1 (L1) are the most numerous TEs in mouse and human genomes. They mobilize via a \u201ccopy and paste\u201d mechanism that requires an RNA intermediate. Although most L1s have lost their activity during evolution, the remaining subset continues to move both in the germline and in adult somatic tissues. Mounting evidence suggests that L1s are active in somatic cells of the mammalian brain and that dysregulated activation of L1s is associated with neuropathology, such as schizophrenia, Rett syndrome and Ataxia telengectasia. Huntington\u2019s disease (HD) is an autosomal dominant disorder that manifests in mid-life and is characterized by neuronal loss, prominently in striatum and deep layers of the cerebral cortex. Typical HD-associated phenotypes include somatic genomic instability, epigenetic and transcriptional dysregulations, impaired neurogenesis and altered DNA damage response. At the same time, in HD, the origin and the role of many genetic and epigenetic modifiers acting on disease onset and progression remain largely unknown. In this scenario, I investigated whether L1 retrotransposition might be altered in HD and if it could have a role in HD pathogenesis. To address this question, using a novel Taqman qPCR technique, I characterized endogenous L1 retrotransposition events in the brains of a precise genetic mouse model of HD, considering both pre-symptomatic and symptomatic developmental stages. From this study, I showed that similar levels of L1 genomic copies are present between HD and control brains. Moreover, differences in full length L1 transcript levels have been reported in HD brains. Interestingly, in HD striatum, at 12 months of age, expression of full length L1s was consistently impaired, whereas in the cortex, L1 mRNA levels were increased in HD mice at 3 months and 24 months of age. The dysregulation of L1 expression in the striatum of 12 months old mice did not appear to be linked to differential deposition of H3K4me3, H3K9me3, H3K27me3 and MeCP2 on L1 promoter in HD conditions. Nonetheless, L1 transcriptional alterations might involve a piRNA-mediated regulation. Indeed, in both cortex and striatum of adult HD and control mice I detected appreciable levels of MILI protein, the crucial factor of piRNA biogenesis, suggesting its role not only in the germline but also in adult mammalian brains, as recently proposed by other two independent works. Additionally, I showed that in a subset of neurons of the adult mouse pre-frontal cortex, endogenous L1 transcription is accompanied by expression of L1-encoded ORF2 protein. Finally, by characterizing the transcription of active murine full length L1 elements in a broad range of developmental stages (from E10 up to 24 months), I described the expression profiles of endogenous L1 elements during the entire mouse development. From this study, I showed that a wave of L1 transcription takes place between E12 and P0 in both striatum and cerebral cortex and it is concomitant with telencephalic neurogenesis. In post-natal stages, L1 transcription is maintained at basal levels

CHARACTERIZATION AND DISTRIBUTION OF NOVEL NON-LTR RETROELEMENTS DRIVING HIGH TELOMERE RFLP DIVERSITY IN CLONAL LINES OF MAGNAPORTHE ORYZAE

The filamentous ascomycete fungus Magnaporthe oryzae is a pathogen of over 50 genera of grasses. Two important diseases it can cause are gray leaf spot in Lolium perenne (perennial ryegrass) and blast in Oryza sativa (rice). The telomeres of M. oryzae isolates causing gray leaf spot are highly variable, and can spontaneously change during fungal culture. In this dissertation, it is shown that a rice-infecting isolate is much more stable at the telomeres than an isolate from gray leaf spot. To determine the molecular basis of telomere instability several gray leaf spot isolates telomeres were cloned, which revealed two non-LTR retrotransposons inserted into the telomere repeats. The elements have been termed Magnaporthe oryzae Telomeric Retrotransposons (MoTeRs). These elements do not have poly-A tails common to many other non-LTR retrotransposons, but instead have telomere like sequences at their 5’ end that allow them to insert into telomeres. Intact copies of MoTeRs were restricted to the telomeres of isolates causing gray leaf spot. Surveys for the presence of these elements in M. oryzae showed they were present in several host-specialized forms including gray leaf spot isolates, but were largely absent in the rice blast isolates. The absence of MoTeRs in rice blast isolates, which are relatively stable by comparison, suggested that the telomere instability in gray leaf spot isolates could be due to MoTeRs. Analyzing spontaneous alterations in telomere restriction fragment profiles of asexual progeny revealed that MoTeRs were involved. Expansion and contraction of MoTeR arrays were observed and account for some telomere restriction profile changes. New telomere formation in asexual progeny followed by MoTeR addition was also observed. Based on this evidence, MoTeRs are largely responsible for the high variability of telomere restriction profiles observed in GLS isolates

Preferential Occupancy of R2 Retroelements on the B Chromosomes of the Grasshopper Eyprepocnemis plorans

R2 non-LTR retrotransposons exclusively insert into the 28S rRNA genes of their host, and are expressed by co-transcription with the rDNA unit. The grasshopper Eyprepocnemis plorans contains transcribed rDNA clusters on most of its A chromosomes, as well as non-transcribed rDNA clusters on the parasitic B chromosomes found in many populations. Here the structure of the E. plorans R2 element, its abundance relative to the number of rDNA units and its retrotransposition activity were determined. Animals screened from five populations contained on average over 12,000 rDNA units on their A chromosomes, but surprisingly only about 100 R2 elements. Monitoring the patterns of R2 insertions in individuals from these populations revealed only low levels of retrotransposition. The low rates of R2 insertion observed in E. plorans differ from the high levels of R2 insertion previously observed in insect species that have many fewer rDNA units. It is proposed that high levels of R2 are strongly selected against in E. plorans, because the rDNA transcription machinery in this species is unable to differentiate between R2-inserted and uninserted units. The B chromosomes of E. plorans contain an additional 7,000 to 15,000 rDNA units, but in contrast to the A chromosomes, from 150 to over 1,500 R2 elements. The higher concentration of R2 in the inactive B chromosomes rDNA clusters suggests these chromosomes can act as a sink for R2 insertions thus further reducing the level of insertions on the A chromosomes. These studies suggest an interesting evolutionary relationship between the parasitic B chromosomes and R2 elements.This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnología (CGL2009-11917) and Plan Andaluz de Investigacion (CVI-6649), and was partially performed by FEDER funds and a grant from the National Institutes of Health (GM42790)

Transposable elements in arthropods genomes with non-canonical reproductive strategies.

Gli elementi trasponibili (TEs) sono componenti universali dei genomi di tutti gli esseri viventi. L'attività ed il movimento degli TEs hanno importanti effetti sul genoma ospite e per questo sono considerati dei fattori importanti nell'evoluzione del genoma. Le relazioni che intercorrono tra TEs e il loro genoma ospite sono ancora ampiamente dibattute. Hickey già nel 1982 suggerì che queste relazioni potevano essere influenzate dalla modalità di riproduzione del genoma ospite. Organismi bisessuali, attraverso i meccanismi propri della meiosi, riuscirebbero a contrastare la proliferazione degli TEs, al contrario negli organismi unisessuali, proprio a causa della mancanza di questi meccanismi, gli TEs tendono a proliferare ed accumulare all'interno del genoma. Al fine di valutare queste ipotesi, ho isolato e caratterizzato gli TEs in organismi con strategie riproduttive non canoniche. Ho condotto le mie analisi in due organismi: gli insetti stecco del genere Bacillus e nel fossile vivente Triops cancriformis. Entrambi presentano strategie riproduttive che vanno dal gonocorismo alla partenogenesi, rendendoli un eccellente modello per lo studio e la caratterizzazione degli TEs. Nel genere Bacillus mi sono focalizzata sullo studio del retrotransposone non-LTR R2. Ho isolato e caratterizzato sette elementi R2 completi; sia in specie gonocoriche che partenogenetiche. Ho trovato un elemento R2 degenerato presente nel genoma di B. rossius da almeno 5 milioni di anni. In oltre, le mie analisi suggeriscono per la prima volta che anche gli elementi R2 possono trasferirsi attraverso eventi di trasferimento orizzontale. I miei dati mostrano una panoramica della composizione di TEs nel genoma di una popolazione partenogenetica di Triops cancriformis. Dalle mie analisi si evince che il 20% della library di triops è composta da TEs.Transposable elements (TEs) are universal components of all living organisms. TEs activity and movement have profound effects on host genome and today many researchers agree to consider them as important actors in genome evolution. The relationships between TEs and their host genomes are still under debate. Different hypotheses were proposed to explain TEs dynamics in host genome; one of these propose that host reproductive strategies can influence TEs evolutionary dynamics (Hickey, 1982). In fact, bisexual organisms, through homologous chromosomes recombination and reassortment during meiosis and amphimixis, can control the spread and proliferation of mobile elements, while unisexual organisms would experience an increase of these elements density due to the inability to eliminate them through exclusive mechanisms of sexual reproduction. In order to evaluate these hypothesis, I isolated and characterized TEs in organisms with non-canonical reproductive strategies. I performed my analyses in two organisms: the stick insects of the Bacillus genus and in the tadpole shrimps T. cancriformis. In both instances reproductive strategies range from bisexual gonochoric reproduction, to unisexual parthenogenesis, making them an excellent model for the study and characterization of TEs. In the Bacillus genus I focused on the R2 non-LTR retrotranposon. I isolated and sequenced by primer walking seven R2 complete elements; both gonochoric and parthenogenetic Bacillus species. I found a R2 degenerate element present in the B. rossius genome since 5 Myr ago. In addition, my analyses for the first time suggest that also R2 retrotransposons can use horizontal transfer as a strategy to colonize a new genome. My data showed a TEs overview in a T. cancriformis parthenogenetic population. In contrast with the theoretical, my analyses highlighted that the 20% of the library is composed by TEs, in which both TEs classes are widely represented (class I, 11.4% and class II, 8.9%)