Sequencing,identification and mapping of primed L1 elements (SIMPLE) reveals significant variation in full length L1 elements between individuals

Background

There are over a half a million copies of L1 retroelements in the human genome which are responsible for as much as 0.5% of new human genetic diseases. Most new L1 inserts arise from young source elements that are polymorphic in the human genome. Highly active polymorphic “hot” L1 source elements have been shown to be capable of extremely high levels of mobilization and result in numerous instances of disease. Additionally, hot polymorphic L1s have been described to be highly active within numerous cancer genomes. These hot L1s result in mutagenesis by insertion of new L1 copies elsewhere in the genome, but also have been shown to generate additional full length L1 insertions which are also hot and able to further retrotranspose. Through this mechanism, hot L1s may amplify within a tumor and result in a continued cycle of mutagenesis.

Results and conclusions

We have developed a method to detect full-length, polymorphic L1 elements using a targeted next generation sequencing approach, Sequencing Identification and Mapping of Primed L1 Elements (SIMPLE). SIMPLE has 94% sensitivity and detects nearly all full-length L1 elements in a genome. SIMPLE will allow researchers to identify hot mutagenic full-length L1s as potential drivers of genome instability. Using SIMPLE we find that the typical individual has approximately 100 non-reference, polymorphic L1 elements in their genome. These elements are at relatively low population frequencies relative to previously identified polymorphic L1 elements and demonstrate the tremendous diversity in potentially active L1 elements in the human population.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1374-y) contains supplementary material, which is available to authorized users.     

Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity

The ubiquity of mobile elements in mammalian genomes poses considerable challenges for the maintenance of genome integrity. The predisposition of mobile elements towards participation in genomic rearrangements is largely a consequence of their interspersed homologous nature. As tracts of nonallelic sequence homology, they have the potential to interact in a disruptive manner during both meiotic recombination and DNA repair processes, resulting in genomic alterations ranging from deletions and duplications to large-scale chromosomal rearrangements. Although the deleterious effects of transposable element (TE) insertion events have been extensively documented, it is arguably through post-insertion genomic instability that they pose the greatest hazard to their host genomes. Despite the periodic generation of important evolutionary innovations, genomic alterations involving TE sequences are far more frequently neutral or deleterious in nature. The potentially negative consequences of this instability are perhaps best illustrated by the >25 human genetic diseases that are attributable to TE-mediated rearrangements. Some of these rearrangements, such as those involving the MLL locus in leukemia and the LDL receptor in familial hypercholesterolemia, represent recurrent mutations that have independently arisen multiple times in human populations. While TE-instability has been a potent force in shaping eukaryotic genomes and a significant source of genetic disease, much concerning the mechanisms governing the frequency and variety of these events remains to be clarified. Here we survey the current state of knowledge regarding the mechanisms underlying mobile element-based genetic instability in mammals. Compared to simpler eukaryotic systems, mammalian cells appear to have several modifications to their DNA-repair ensemble that allow them to better cope with the large amount of interspersed homology that has been generated by TEs. In addition to the disruptive potential of nonallelic sequence homology, we also consider recent evidence suggesting that the endonuclease products of TEs may also play a key role in instigating mammalian genomic instability.     

Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes

Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl₂ favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl₂ doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the “error prone” non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.     

Individualizing kinase-targeted cancer therapy: the paradigm of chronic myeloid leukemia

The success of tyrosine kinase inhibitors in treating chronic myeloid leukemia highlights the potential of targeting oncogenic kinases with small molecules. By using drug activity profiles and individual patient genotypes, one can guide personalized therapy selection for patients with resistance.     

ERK2, but Not ERK1, Mediates Acquired and “De novo” Resistance to Imatinib Mesylate: Implication for CML Therapy

Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation.     

Down-regulation of hypusine biosynthesis in plasmodium by inhibition of S-adenosyl-methionine-decarboxylase

An important issue facing global health today is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries. Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance, cost and the current lack of innovations in medicinal chemistry. Depletion of polyamines in the intraerythrocytic phase of P. falciparum is a promising strategy for the development of new antimalarials since intracellular levels of putrescine, spermidine and spermine are increased during cell proliferation. S-adenosyl-methionine-decarboxylase (AdoMETDC) is a key enzyme in the biosynthesis of spermidine. The AdoMETDC inhibitor CGP 48664A, known as SAM486A, inhibited the separately expressed plasmodial AdoMETDC domain with a Km _i of 3 μM resulting in depletion of spermidine. Spermidine is an important precursor in the biosynthesis of hypusine. This prompted us to investigate a downstream effect on hypusine biosynthesis after inhibition of AdoMETDC. Extracts from P. falciparum in vitro cultures that were treated with 10 μM SAM 486A showed suppression of eukaryotic initiation factor 5A (eIF-5A) in comparison to the untreated control in two-dimensional gel electrophoresis. Depletion of eIF-5A was also observed in Western blot analysis with crude protein extracts from the parasite after treatment with 10 μM SAM486A. A determination of the intracellular polyamine levels revealed an approximately 27% reduction of spemidine and a 75% decrease of spermine while putrescine levels increased to 36%. These data suggest that inhibition of AdoMetDc provides a novel strategy for eIF-5A suppression and the design of new antimalarials.