Reviving a 60 million year old LINE-1 element.

Mobile elements have significantly impacted genome structure of most organisms. The continued activity of the mobile element, LINE-1 (L1), through time has contributed to the accumulation of over half a million L1 copies in the human genome. Most copies in the human genome belong to evolutionary older extinct L1s. Here we apply our previous published approach to "revive" the extinct L1 PA13A; an L1 family that was active about 60 million year ago (mya). The reconstructed L1PA13A is retrocompentent in culture, but shows a significantly lower level of activity in HeLa cells when compared to the modern L1 element (L1PA1) and a 40 million year old L1PA8. L1 elements code for two proteins (ORF1p and ORF2p) that are necessary for retrotransposition. Using PA13A-PA1 and PA13A-PA8 L1 chimeric elements, we determined that both the ORF1p and ORF2p contribute to the observed decrease in retrotransposition efficiency of L1PA13A. The lower retrotransposition rate of L1PA13A is consistent in both human and rodent cell lines. However, in rodent cells, the chimeric element L1PA:1-13 containing the modern L1PA1 ORF1p shows a recovery in the retrotransposition rate, suggestive that the L1PA13A ORF2p efficiently drives retrotransposition in these cells. The functionality of the L1PA13A ORF2p was further confirmed by demonstrating its ability to drive Alu retrotransposition in rodent cells. The variation in L1PA13A retrotransposition rates observed between rodent and human cells are suggestive that cellular environment significantly affects retrotransposition efficiency, which may be mediated through an interaction with ORF1p. Based on these observations, we speculate that the observed differences between cell lines may reflect an evolutionary adaptation of the L1 element to its host cell.

The role of Alu-derived RNAs in Alzheimer's and other neurodegenerative conditions.

Non-coding RNAs have emerged as essential contributors to neuroinflammation. The Alu element is the most abundant potential source of non-coding RNA in the human genome represented by over 1.1 million copies totaling ∼10% of the genome's mass. Accumulation of "Alu RNA" was observed in the brains of individuals with dementia and Creutzfeldt-Jakob disease - a degenerative brain disorder. "Alu RNAs" activate inflammatory pathways and apoptosis in the non-neural cells. In particular, the "Alu RNA" cytotoxicity is suggested as a mechanism in retinal pigment epithelium (RPE), a compartment damaged in the process of age-related macular degeneration. In RPE cells, the deficiency of Dicer is reported to lead to an accumulation of P3Alu transcripts, subsequent activation of the ERK1/2 signaling pathway, and the formation of NLRP3 inflammasome. In turn, these events result in RPE cell death by apoptosis. Importantly, RPE cells are of neuroectodermal origin, these cells display more similarity to neurons than to other epithelial cells. Thus, it is plausible that the mechanisms of "Alu RNA" cytotoxicity in brain neurons are similar to that in RPE. We hypothesize that accumulation of polymerase III-transcribed noncoding RNA of Alu (P3Alu) may contribute to both neuroinflammation and neurodegeneration associated with Alzheimer's disease (AD) and other degenerative brain disorders. This hypothesis points toward a novel molecular pathway not previously considered for the treatment of AD.

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus. Although in some instances retrotransposition efficiencies slightly diminished, all fusion proteins containing an intact ORF2 were capable of driving retrotransposition. Second, the stability of individual ORF2 fusion proteins varies and difficult to predict. Third, DBDs that require the formation of multimers for target recognition are unlikely to modify targeting of ORF2p-driven insertions. Fourth, the more components needed to assemble into a complex to drive targeted retrotransposition, the less likely the strategy will increase targeted insertions. Fifth, abundance of target sequences present in the genome will likely dictate the effectiveness and efficiency of targeted insertions. Lastly, the cleavage capabilities of Cas9 (or a Cas9 nickase variant) are unable to substitute for the L1 ORF2 endonuclease domain functions, suggestive that the endonuclease domain has alternate functions needed for retrotransposition. From these studies, we conclude that the most critical component for the modification of the human L1 ORF2 protein to drive targeted insertions is the selection of the DBD due to the varying functional requirements and impacts on protein stability.

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition.

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a "copy-and-paste" mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3' DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events.

Meeting report for Odd Pols 2016: Ann Arbor 2.0.

The Tenth International Conference on Transcription by RNA Polymerases I, III, IV and V (the 'Odd Pols') was held June 24-28, 2016 at the University of Michigan, Ann Arbor, USA and organized by David Engelke, Deborah Johnson, Richard Maraia, Lawrence Rothblum, David Schneider, Andrzej Wierzbicki and Astrid Engel. The organizers are grateful for the support from the Rackham Graduate School of the University of Michigan for providing the meeting venue. The environment provided a great background with unexpected encounters with fireflies, free live music and a festive fireworks display. The meeting was composed of eleven oral sessions and a poster session. The keynote speaker, Dave Engelke, opened the meeting with his presentation entitled "A personal history of pol III transcription - how we got here from the 'good old days'." The meeting drew attendees from sixteen countries that shared their research discoveries. Here we present some of the highlights from the meeting using summaries provided by the participants.

Carcinogenic effects of oil dispersants: A KEGG pathway-based RNA-seq study of human airway epithelial cells.

The health impacts of the BP oil spill are yet to be further revealed as the toxicological effects of oil products and dispersants on human respiratory system may be latent and complex, and hence difficult to study and follow up. Here we performed RNA-seq analyses of a system of human airway epithelial cells treated with the BP crude oil and/or dispersants Corexit 9500 and Corexit 9527 that were used to help break up the oil spill. Based on the RNA-seq data, we then systemically analyzed the transcriptomic perturbations of the cells at the KEGG pathway level using two pathway-based analysis tools, GAGE (generally applicable gene set enrichment) and GSNCA (Gene Sets Net Correlations Analysis). Our results suggested a pattern of change towards carcinogenesis for the treated cells marked by upregulation of ribosomal biosynthesis (hsa03008) (p=1.97E-13), protein processing (hsa04141) (p=4.09E-7), Wnt signaling (hsa04310) (p=6.76E-3), neurotrophin signaling (hsa04722) (p=7.73E-3) and insulin signaling (hsa04910) (p=1.16E-2) pathways under the dispersant Corexit 9527 treatment, as identified by GAGE analysis. Furthermore, through GSNCA analysis, we identified gene co-expression changes for several KEGG cancer pathways, including small cell lung cancer pathway (hsa05222, p=9.99E-5), under various treatments of oil/dispersant, especially the mixture of oil and Corexit 9527. Overall, our results suggested carcinogenic effects of dispersants (in particular Corexit 9527) and their mixtures with the BP crude oil, and provided further support for more stringent safety precautions and regulations for operations involving long-term respiratory exposure to oil and dispersants.

Effects of corexit oil dispersants and the WAF of dispersed oil on DNA damage and repair in cultured human bronchial airway cells, BEAS-2B.

Large quantities of dispersants were used as a method to disperse the roughly 210 million gallons of spilled crude oil that consumed the Gulf of Mexico. Little is known if the oil-dispersant and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to Corexit dispersants EC9500 and EC9527, Water Accommodated Fraction (WAF) -crude, WAF-9500 + Oil, and WAF-9527 + Oil. Cellular cytotoxicity to WAF-dispersed oil samples was observed at concentrations greater than 1000 ppm with over 70% of observed cellular death. At low concentration exposures (100 and 300 ppm) DNA damage was evidenced by the detection of single strand breaks (SSBs) and double strand breaks (DSBs) as measured by alkaline and neutral comet assay analyses. Immunoblot analyses of the phosphorylated histone H2A.X (É£-H2A.X) and tumor suppressor p53 protein confirmed activation of the DNA damage response due to the exposure-induced DNA breaks. Although, many xenobiotics interfere with DNA repair pathways, in vitro evaluation of the nucleotide excision repair (NER) and DSB repair pathways appear to be unaffected by the oil-dispersant mixtures tested. Overall, this study supports that oil-dispersant mixtures induce genotoxic effects in culture.

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.

SINE Retrotransposition: Evaluation of Alu Activity and Recovery of De Novo Inserts.

Mobile element activity is of great interest due to its impact on genomes. However, the types of mobile elements that inhabit any given genome are remarkably varied. Among the different varieties of mobile elements, the Short Interspersed Elements (SINEs) populate many genomes, including many mammalian species. Although SINEs are parasites of Long Interspersed Elements (LINEs), SINEs have been highly successful in both the primate and rodent genomes. When comparing copy numbers in mammals, SINEs have been vastly more successful than other nonautonomous elements, such as the retropseudogenes and SVA. Interestingly, in the human genome the copy number of Alu (a primate SINE) outnumbers LINE-1 (L1) copies 2 to 1. Estimates suggest that the retrotransposition rate for Alu is tenfold higher than LINE-1 with about 1 insert in every twenty births. Furthermore, Alu-induced mutagenesis is responsible for the majority of the documented instances of human retroelement insertion-induced disease. However, little is known on what contributes to these observed differences between SINEs and LINEs. The development of an assay to monitor SINE retrotransposition in culture has become an important tool for the elucidation of some of these differences. In this chapter, we present details of the SINE retrotransposition assay and the recovery of de novo inserts. We also focus on the nuances that are unique to the SINE assay.

The impact of oil spill to lung health--Insights from an RNA-seq study of human airway epithelial cells.

The Deepwater Horizon oil spill (BP oil spill) in the Gulf of Mexico was a unique disaster event, where a huge amount of oil spilled from the sea bed and a large volume of dispersants were applied to clean the spill. The operation lasted for almost 3 months and involved >50,000 workers. The potential health hazards to these workers may be significant as previous research suggested an association of persistent respiratory symptoms with exposure to oil and oil dispersants. To reveal the potential effects of oil and oil dispersants on the respiratory system at the molecular level, we evaluated the transcriptomic profile of human airway epithelial cells grown under treatment of crude oil, the dispersants Corexit 9500 and Corexit 9527, and oil-dispersant mixtures. We identified a very strong effect of Corexit 9500 treatment, with 84 genes (response genes) differentially expressed in treatment vs. control samples. We discovered an interactive effect of oil-dispersant mixtures; while no response gene was found for Corexit 9527 treatment alone, cells treated with Corexit 9527+oil mixture showed an increased number of response genes (46 response genes), suggesting a synergic effect of 9527 with oil on airway epithelial cells. Through GO (gene ontology) functional term and pathway-based analysis, we identified upregulation of gene sets involved in angiogenesis and immune responses and downregulation of gene sets involved in cell junctions and steroid synthesis as the prevailing transcriptomic signatures in the cells treated with Corexit 9500, oil, or Corexit 9500+oil mixture. Interestingly, these key molecular signatures coincide with important pathological features observed in common lung diseases, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Our study provides mechanistic insights into the detrimental effects of oil and oil dispersants to the respiratory system and suggests significant health impacts of the recent BP oil spill to those people involved in the cleaning operation.

Altering Genomic Integrity: Heavy Metal Exposure Promotes Transposable Element-Mediated Damage.

Maintenance of genomic integrity is critical for cellular homeostasis and survival. The active transposable elements (TEs) composed primarily of three mobile element lineages LINE-1, Alu, and SVA comprise approximately 30% of the mass of the human genome. For the past 2 decades, studies have shown that TEs significantly contribute to genetic instability and that TE-caused damages are associated with genetic diseases and cancer. Different environmental exposures, including several heavy metals, influence how TEs interact with its host genome increasing their negative impact. This mini-review provides some basic knowledge on TEs, their contribution to disease, and an overview of the current knowledge on how heavy metals influence TE-mediated damage.

Meeting report for "OddPols" 2014: the odds invite an even.

The Ninth International Biennial Conference on RNA Polymerases I and III (the "OddPols") was held on June 19-21, 2014 at the University of Michigan, Ann Arbor, USA. Sponsored by New England Biolabs, the Cayman Chemical Company, the Rackham Graduate School and the University of Michigan Health System, and organized by David Engelke, Craig Pikaard, Lawrence Rothblum, Andrzej Wierzbicki and Astrid Engel. This year at the conference, the "odds" were increased by expanding the usual topics on the advances in RNA polymerases I and III research to include presentations on RNA polymerase IV and V. The keynote speaker, Craig Pikaard, opened the meeting with his presentation entitled "Five nuclear multisubunit RNA polymerases". The meeting drew attendees from fourteen countries that shared their research discoveries through oral and poster presentations. The talks were organized into 11 sessions covering seven distinct topics. Here we present some of the highlights from the meeting using summaries provided by the participants.

Alu elements: an intrinsic source of human genome instability.

Alu elements are ∼300bp sequences that have amplified via an RNA intermediate leading to the accumulation of over 1 million copies in the human genome. Although a few of the copies are active, Alu germline activity is the highest of all human retrotransposons and does significantly contribute to genetic disease and population diversity. There are two basic mechanisms by which Alu elements contribute to disease: through insertional mutagenesis and as a large source of repetitive sequences that contribute to nonallelic homologous recombination (NAHR) that cause genetic deletions and duplications.

Effects of COREXIT dispersants on cytotoxicity parameters in a cultured human bronchial airway cells, BEAS-2B.

The objective of this study was to assess the cytotoxicity of COREXIT dispersants EC9500A, EC9527A, and EC9580A on human airway BEAS-2B epithelial cells. Cells were exposed to dispersants for 2 or 24 h at concentrations ranging from 0 to 300 ppm. COREXIT EC9527 at 100 ppm produced 50% viability loss as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 24 h. COREXIT 9527 at 200 ppm produced 50% cell death at 2 h and 100% at 24 h. At 300 ppm COREXIT 9527 induced 100% cell death at 2 or 24 h. In the case of COREXIT 9500A 50% cell viability was noted with 200 ppm at 2 or 24 h, with a significant decrease in cell survival to 2% at 300 ppm. In contrast, no marked change in cell viability was observed in cells treated at any COREXIT 9580A concentration examined. Western blot analysis showed an increase in expression of LC3B, a marker of autophagy, in cells treated for 2 h with 300 ppm COREXIT EC9527A as well as 100 or 300 ppm Corexit EC9500A. No marked effect on LC3B expression was observed for any COREXIT 9580A concentration. Apoptosis markers as measured by cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were detectable only in cells incubated with 300 ppm COREXIT EC9527A. Although all three dispersants induced enhanced generation of reactive oxygen species (ROS) after 2-h treatment at 300 ppm, Western blot analysis revealed that 2-h incubation was not sufficient to induce a significant change in the protein expression of superoxide dismutases SOD1, SOD2, and SOD3. Data thus indicate exposure to certain dispersants may be harmful to human airway epithelial cells in a concentration-dependent manner.

Meeting report for Odd Pols 2012.

The Eighth International Biennial Conference on RNA polymerases I and III (the 'Odd Pols') was held June 7-11, 2012 at The Airlie Center in Warrenton Virginia, USA. It was sponsored by the Universite Laval and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, and organized by Rich Maraia and Tom Moss. The meeting honored the memory of Pierre Thuriaux (Jan 1, 1950-March 18, 2012) and David Schneider reminisced on the important accomplishments his mentor Masayasu Nomura (1927-2011). The goal of the conference was to bring together the world's experts on RNA polymerase I and RNA polymerase III to highlight and share their latest results and varied experimental approaches. The meeting drew attendees from twelve countries and most contributed through oral and poster presentations. The talks were organized into several sessions subdivided into 10 distinct topics. The keynote speaker, Ian Willis, opened the meeting with his presentation entitled "New Regulators of Signaling to Odd Pols" and the closing presentation was given by Patrick Cramer with his presentation "Conservation of the RNA polymerase I, II and III transcription initiation machineries". Here we present some of the highlights from the meeting using summaries provided by the participants.

Molecular reconstruction of extinct LINE-1 elements and their interaction with nonautonomous elements.

Non-long terminal repeat retroelements continue to impact the human genome through cis-activity of long interspersed element-1 (LINE-1 or L1) and trans-mobilization of Alu. Current activity is dominated by modern subfamilies of these elements, leaving behind an evolutionary graveyard of extinct Alu and L1 subfamilies. Because Alu is a nonautonomous element that relies on L1 to retrotranspose, there is the possibility that competition between these elements has driven selection and antagonistic coevolution between Alu and L1. Through analysis of synonymous versus nonsynonymous codon evolution across L1 subfamilies, we find that the C-terminal ORF2 cys domain experienced a dramatic increase in amino acid substitution rate in the transition from L1PA5 to L1PA4 subfamilies. This observation coincides with the previously reported rapid evolution of ORF1 during the same transition period. Ancestral Alu sequences have been previously reconstructed, as their short size and ubiquity have made it relatively easy to retrieve consensus sequences from the human genome. In contrast, creating constructs of extinct L1 copies is a more laborious task. Here, we report our efforts to recreate and evaluate the retrotransposition capabilities of two ancestral L1 elements, L1PA4 and L1PA8 that were active ~18 and ~40 Ma, respectively. Relative to the modern L1PA1 subfamily, we find that both elements are similarly active in a cell culture retrotransposition assay in HeLa, and both are able to efficiently trans-mobilize Alu elements from several subfamilies. Although we observe some variation in Alu subfamily retrotransposition efficiency, any coevolution that may have occurred between LINEs and SINEs is not evident from these data. Population dynamics and stochastic variation in the number of active source elements likely play an important role in individual LINE or SINE subfamily amplification. If coevolution also contributes to changing retrotransposition rates and the progression of subfamilies, cell factors are likely to play an important mediating role in changing LINE-SINE interactions over evolutionary time.

Rescuing Alu: recovery of new inserts shows LINE-1 preserves Alu activity through A-tail expansion.

Alu elements are trans-mobilized by the autonomous non-LTR retroelement, LINE-1 (L1). Alu-induced insertion mutagenesis contributes to about 0.1% human genetic disease and is responsible for the majority of the documented instances of human retroelement insertion-induced disease. Here we introduce a SINE recovery method that provides a complementary approach for comprehensive analysis of the impact and biological mechanisms of Alu retrotransposition. Using this approach, we recovered 226 de novo tagged Alu inserts in HeLa cells. Our analysis reveals that in human cells marked Alu inserts driven by either exogenously supplied full length L1 or ORF2 protein are indistinguishable. Four percent of de novo Alu inserts were associated with genomic deletions and rearrangements and lacked the hallmarks of retrotransposition. In contrast to L1 inserts, 5' truncations of Alu inserts are rare, as most of the recovered inserts (96.5%) are full length. De novo Alus show a random pattern of insertion across chromosomes, but further characterization revealed an Alu insertion bias exists favoring insertion near other SINEs, highly conserved elements, with almost 60% landing within genes. De novo Alu inserts show no evidence of RNA editing. Priming for reverse transcription rarely occurred within the first 20 bp (most 5') of the A-tail. The A-tails of recovered inserts show significant expansion, with many at least doubling in length. Sequence manipulation of the construct led to the demonstration that the A-tail expansion likely occurs during insertion due to slippage by the L1 ORF2 protein. We postulate that the A-tail expansion directly impacts Alu evolution by reintroducing new active source elements to counteract the natural loss of active Alus and minimizing Alu extinction.

Alu expression in human cell lines and their retrotranspositional potential.

BACKGROUND: The vast majority of the 1.1 million Alu elements are retrotranspositionally inactive, where only a few loci referred to as 'source elements' can generate new Alu insertions. The first step in identifying the active Alu sources is to determine the loci transcribed by RNA polymerase III (pol III). Previous genome-wide analyses from normal and transformed cell lines identified multiple Alu loci occupied by pol III factors, making them candidate source elements. FINDINGS: Analysis of the data from these genome-wide studies determined that the majority of pol III-bound Alus belonged to the older subfamilies Alu S and Alu J, which varied between cell lines from 62.5% to 98.7% of the identified loci. The pol III-bound Alus were further scored for estimated retrotransposition potential (ERP) based on the absence or presence of selected sequence features associated with Alu retrotransposition capability. Our analyses indicate that most of the pol III-bound Alu loci candidates identified lack the sequence characteristics important for retrotransposition. CONCLUSIONS: These data suggest that Alu expression likely varies by cell type, growth conditions and transformation state. This variation could extend to where the same cell lines in different laboratories present different Alu expression patterns. The vast majority of Alu loci potentially transcribed by RNA pol III lack important sequence features for retrotransposition and the majority of potentially active Alu loci in the genome (scored high ERP) belong to young Alu subfamilies. Our observations suggest that in an in vivo scenario, the contribution of Alu activity on somatic genetic damage may significantly vary between individuals and tissues.