Alu Subfamilies Research Articles

Profiling of repetitive RNA sequences in the blood plasma of patients with cancer

Liquid biopsies provide a means for the profiling of cell-free RNAs secreted by cells throughout the body. Although well-annotated coding and non-coding transcripts in blood are readily detectable and can serve as biomarkers of disease, the overall diagnostic utility of the cell-free transcriptome remains unclear. Here we show that RNAs derived from transposable elements and other repeat elements are enriched in the cell-free transcriptome of patients with cancer, and that they serve as signatures for the accurate classification of the disease. We used repeat-element-aware liquid-biopsy technology and single-molecule nanopore sequencing to profile the cell-free transcriptome in plasma from patients with cancer and to examine millions of genomic features comprising all annotated genes and repeat elements throughout the genome. By aggregating individual repeat elements to the subfamily level, we found that samples with pancreatic cancer are enriched with specific Alu subfamilies, whereas other cancers have their own characteristic cell-free RNA profile. Our findings show that repetitive RNA sequences are abundant in blood and can be used as disease-specific diagnostic biomarkers.

Framework of the Alu Subfamily Evolution in the Platyrrhine Three-Family Clade of Cebidae, Callithrichidae, and Aotidae.

The history of Alu retroposons has been choreographed by the systematic accumulation of inherited diagnostic nucleotide substitutions to form discrete subfamilies, each having a distinct nucleotide consensus sequence. The oldest subfamily, AluJ, gave rise to AluS after the split between Strepsirrhini and what would become Catarrhini and Platyrrhini. The AluS lineage gave rise to AluY in catarrhines and to AluTa in platyrrhines. Platyrrhine Alu subfamilies Ta7, Ta10, and Ta15 were assigned names based on a standardized nomenclature. However, with the subsequent intensification of whole genome sequencing (WGS), large scale analyses to characterize Alu subfamilies using the program COSEG identified entire lineages of subfamilies simultaneously. The first platyrrhine genome with WGS, the common marmoset (Callithrix jacchus; [caljac3]), resulted in Alu subfamily names sf0 to sf94 in an arbitrary order. Although easily resolved by alignment of the consensus sequences, this naming convention can become increasingly confusing as more genomes are independently analyzed. In this study, we reported Alu subfamily characterization for the platyrrhine three-family clade of Cebidae, Callithrichidae, and Aotidae. We investigated one species/genome from each recognized family of Callithrichidae and Aotidae and of both subfamilies (Cebinae andSaimiriinae) of the family Cebidae. Furthermore, we constructed a comprehensive network of Alu subfamily evolution within the three-family clade of platyrrhines to provide a working framework for future research. Alu expansion in the three-family clade has been dominated by AluTa15 and its derivatives.

Genome-wide perturbations of Alu expression and Alu-associated post-transcriptional regulations distinguish oligodendroglioma from other gliomas

Alu is a primate-specific repeat element in the human genome and has been increasingly appreciated as a regulatory element in many biological processes. But the appreciation of Alu has been limited in tumorigenesis, especially for brain tumor. To investigate the relevance of Alu to the gliomagenesis, we studied Alu element-associated post-transcriptional processes and the RNA expression of the element by performing RNA-seq for a total of 41 pairs of neurotypical and diverse glioma brain tissues. We find that A-to-I editing and circular RNA levels, as well as Alu RNA expression, are decreased overall in gliomas, compared to normal tissue. Interestingly, grade 2 oligodendrogliomas are least affected in A-to-I editing and circular RNA levels among gliomas, whereas they have a higher proportion of down-regulated Alu subfamilies, compared to the other gliomas. These findings collectively imply a unique pattern of Alu-associated transcriptomes in grade 2 oligodendroglioma, providing an insight to gliomagenesis from the perspective of an evolutionary genetic element.

Aluelement in the RNA binding motif protein, X-linked 2 (RBMX2) gene found to be linked to bipolar disorder.

ObjectiveWe have used long-read single molecule, real-time (SMRT) sequencing to fully characterize a ~12Mb genomic region on chromosome Xq24-q27, significantly linked to bipolar disorder (BD) in an extended family from a genetic sub-isolate. This family segregates BD in at least four generations with 24 affected individuals.MethodsWe selected 16 family members for targeted sequencing. The selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed hybrid capture probes enriching for 5-9Kb fragments spanning the entire 12Mb region that were then sequenced to screen for candidate structural variants (SVs) that could explain the increased risk for BD in this extended family.ResultsAltogether, 201 variants were detected in the critically linked region. Although most of these represented common variants, three variants emerged that showed near-perfect segregation among all BD type I affected individuals. Two of the SVs were identified in or near genes belonging to the RNA Binding Motif Protein, X-Linked (RBMX) gene family—a 330bp Alu (subfamily AluYa5) deletion in intron 3 of the RBMX2 gene and an intergenic 27bp tandem repeat deletion between the RBMX and G protein-coupled receptor 101 (GPR101) genes. The third SV was a 50bp tandem repeat insertion in intron 1 of the Coagulation Factor IX (F9) gene.ConclusionsAmong the three genetically linked SVs, additional evidence supported the Alu element deletion in RBMX2 as the leading candidate for contributing directly to the disease development of BD type I in this extended family.

A comprehensive analysis of chimpanzee (Pan Troglodytes)-specific AluYb8 element.

Alu elements are most abundant retrotransposons with > 1.2 million copies in the primate genome. AluYb8 subfamily was diverged from AluY lineage, and has accumulated eight diagnostic mutations and 7-bp duplication during primate evolution. A total of 1851 AluYb copies are present in the human genome, and most of them are human-specific. On the other hand, only a few AluYb8 copies were identified in the chimpanzee genome by previous studies on AluYb8. The significantly different number of species-specific AluYb8 elements between human and chimpanzee might result from the incompletion of chimpanzee reference genome sequences at the time of the previous study. AluYb8 elements could generate genomic structural variations in the chimpanzee genome. This study aimed to identify and characterize chimpanzee-specific AluYb elements using the most updated chimpanzee reference genome sequences (Jan. 2018, panTro6). To identify chimpanzee-specific AluYb8, we carried out genomic comparison with non-chimpanzee primate genome using the UCSC table browser. In addition, chimpanzee-specific AluYb8 candidates were manually inspected and experimentally verified using PCR and Sanger sequencing. Among a total of 231 chimpanzee-specific AluYb8 candidates, 11 of the candidates are chimpanzee-specific AluYb8, and 29 elements are shared between the chimpanzee and non-chimpanzee primate genomes. Through the sequence analysis of AluYb8 and other Alu subfamilies, we were able to observe various diagnostic mutations and variable length duplications in 7-bp duplication region of AluYb8 element. In addition, we further validated two of the chimpanzee-specific AluYb8 elements (CS8 and CS20) that were not previously discovered by display PCR and Sanger sequencing. Interestingly, we identified a AluYb8 insertion-mediated deletion (CS8 locus) in the chimpanzee genome. Our study found that AluYb8 elements are much more abundant in the human genome than chimpanzee genome, and that it could be due to the absence of hyperactive "master" AluYb8 elements in the chimpanzee genome.

The Simons Genome Diversity Project: A Global Analysis of Mobile Element Diversity.

Ongoing retrotransposition of Alu, LINE-1, and SINE–VNTR–Alu elements generates diversity and variation among human populations. Previous analyses investigating the population genetics of mobile element insertions (MEIs) have been limited by population ascertainment bias or by relatively small numbers of populations and low sequencing coverage. Here, we use 296 individuals representing 142 global populations from the Simons Genome Diversity Project (SGDP) to discover and characterize MEI diversity from deeply sequenced whole-genome data. We report 5,742 MEIs not originally reported by the 1000 Genomes Project and show that high sampling diversity leads to a 4- to 7-fold increase in MEI discovery rates over the original 1000 Genomes Project data. As a result of negative selection, nonreference polymorphic MEIs are underrepresented within genes, and MEIs within genes are often found in the transcriptional orientation opposite that of the gene. Globally, 80% of Alu subfamilies predate the expansion of modern humans from Africa. Polymorphic MEIs show heterozygosity gradients that decrease from Africa to Eurasia to the Americas, and the number of MEIs found uniquely in a single individual are also distributed in this general pattern. The maximum fraction of MEI diversity partitioned among the seven major SGDP population groups (FST) is 7.4%, similar to, but slightly lower than, previous estimates and likely attributable to the diverse sampling strategy of the SGDP. Finally, we utilize these MEIs to extrapolate the primary Native American shared ancestry component to back to Asia and provide new evidence from genome-wide identical-by-descent genetic markers that add additional support for a southeastern Siberian origin for most Native Americans.

Sequence Analysis of Homologous Alu Elements: A Novel Methodology for Primate Phylogenetic Tree Construction

Non‐human primates are essential model organisms across a broad spectrum of biomedical research, from drug discovery & development to insights into immunological responses. The study of phylogeny enhances understanding of the particular experimental advantages of each model organism. Despite considerable efforts invested into developing currently accepted practices of phylogenetic analysis, these strategies often involve significant expenditures of time and resources. To address these concerns, we developed a novel computational approach to primate phylogenetic tree construction using the Alu transposable element. Short Interspersed Nuclear Elements such as Alu have previously been validated as accurate tools for phylogenetic analysis, as they predominantly evolve in a unidirectional and homoplasy‐free manner. However, previous Alu‐based investigations into phylogenetic relationships have been limited to detecting specific Alu insertion polymorphisms as a proxy for evolutionary divergence. Here, we present a novel methodology for primate phylogenetic analysis using genetic diversity in homologous Alu sequences. Alu elements for 11 primate genomes, classified by subfamily, were extracted from the University of California Santa Cruz’s Genome Table Browser. Subsequently, multiple sequence alignments were performed to identify which Alu sequences were homologous among the 11 primates. Following identification, phylogenetic trees for each of 22 conserved subfamilies were constructed. These trees were then compared to randomly generated phylogenetic trees and found to be significantly (p < 1e‐10) more accurate on all tree distance metrics calculated (Robinson‐Foulds, Path difference, Subtree Pruning and Regrafting, Hamming distance, and Penny Hendy). The Alu‐based trees were then compared to published primate phylogenetic trees to confirm the validity of this technique. No significant difference in accuracy was found for the most accurate Alu subfamily tested, demonstrating its validity. We therefore propose that our approach of comparing homologous Alu sequences can serve as a supplement or alternative to current primate phylogenetic tree construction.

Analysis of retrotransposon subfamily DNA methylation reveals novel early epigenetic changes in chronic lymphocytic leukemia.

Retrotransposons such as LINE-1 and Alu comprise >25% of the human genome. While global hypomethylation of these elements has been widely reported in solid tumours, their epigenetic dysregulation is yet to be characterised in chronic lymphocytic leukemia (CLL), and there has been scant consideration of their evolutionary history that mediates sensitivity to hypomethylation. Here, we developed an approach for locus- and evolutionary subfamily-specific analysis of retrotransposons using the Illumina Infinium Human Methylation 450K microarray platform, which we applied to publicly-available datasets from CLL and other haematological malignancies. We identified 9,797 microarray probes mapping to 117 LINE-1 subfamilies and 13,130 mapping to 37 Alu subfamilies. Of these, 10,782 were differentially methylated (PFDR<0.05) in CLL patients (n=139) compared with healthy individuals (n=14), with enrichment at enhancers (P=0.002). Differential methylation was associated with evolutionary age of LINE-1 (r2=0.31, P=0.003) and Alu (r2=0.74, P=0.002) elements, with greater hypomethylation of older subfamilies (L1M, AluJ). Locus-specific hypomethylation was associated with differential expression of proximal genes, including DCLK2, HK1, ILRUN, TANK, TBCD, TNFRSF1B and TXNRD2, with higher expression of DCLK2 and TNFRSF1B associated with reduced patient survival. Hypomethylation at nine loci was highly frequent in CLL (>90% patients) but not observed in healthy individuals or other leukaemias, and was detectable in blood samples taken prior to CLL diagnosis in 9 of 82 individuals from the Melbourne Collaborative Cohort Study. Our results demonstrate differential methylation of retrotransposons in CLL by their evolutionary heritage that modulates expression of proximal genes.

Effects of Prenatal Air Pollution on Family-Specific, Genome-Wide, Repetitive Element Methylation in Cord Blood

OPS 48: Air pollution and epigenetics, Room 417, Floor 4, August 26, 2019, 4:30 PM - 5:30 PM Background/Objective: Repetitive elements (REs) comprise more than 50% of the genome and methylation suppresses their transpositions (insertions into other genomic regions) that can damage DNA. Global average RE methylation is inversely associated with particulate matter ≤2.5µm (PM2.5) exposure, however, averaging all RE methylation may overlook their biological activity, as RE subfamilies and individual REs vary in methylation. We employed a method predicting genome-wide, RE subfamily-specific, methylation in cord blood and investigated their associations with prenatal PM2.5 exposure. Methods: Trimester average PM2.5 was estimated using a satellite-based spatio-temporally resolved prediction model and residence location of 443 women from the PROGRESS birth-cohort in Mexico City. We measured cord blood DNA methylation with the Illumina EPICArray using a random forest-based algorithm to predict genome-wide, locus-specific CpG methylation of long interspersed elements-1 (L1) and a short interspersed element (Alu) using nearby CpG sites. We modeled associations between PM2.5 with: 1) methylation of subfamilies within the most recent L1PA, L1PB, and Alu lineages averaged across the genome; and 2) genomic locus-specific aggregates of 2 or more adjacent RE CpGs from these subfamilies. Results: We imputed methylation of 1,183 CpG aggregates and 25 subfamilies within the L1PA and L1PB lineages and 69,596 aggregates and 36 Alu subfamilies with mean methylation of 92%. Average methylation of 20 L1 and 35 Alu subfamilies was inversely associated with 2nd trimester PM2.5. Restricting analysis to full-length L1, methylation of L1PA2, L1PA3, L1PA5, and the active element, L1HS, were inversely associated with PM2.5 in the 2nd and 3rd trimester. Investigations of individual CpGs yielded two aggregates associated with PM2.5 after multiple comparison corrections. Conclusions: Reduced RE subfamily methylation may promote transpositions and cell toxicity that could be related to health endpoints such as cancer. Future work will investigate RE expression and whether RE methylation/expression mediate relationships between PM2.5 exposure and health.

Bioinformatics analysis of Alu components at the level of genome 3D structure

The interphase chromatin is folded in the nucleus in a hierarchical manner, including the nucleosome, the "beads on a string" structure composed of nucleosomes, the solenoid fiber structure, the chromatin/DNA loop structure (chromatin/DNA loop), and the topologically associated domain (TAD). Among them, TAD is considered to be the basic unit of the 3D structure of chromatin because it is relatively stable and conserved in different cell types. Alu elements occupy a large proportion in the mammalian genomes. There are a wide variety of Alu elements, but their functional characterizations are limited to date. This study investigates the role of Alu elements in the assembly of 3D chromatin conformation. The evolutionary process of the Alu subfamily was explored by the distance relationship of the 3D structure of chromatin. We found that the proportion of Alu elements in high-density chromatin interaction increased with higher similarity, indicating that Alu plays an important role in the construction of chromatin 3D structure. There is a certain positive correlation between the strength of the upper interaction and the evolutionary relationship. In sum, the Alu elements with relatively close distances in the 1D sequence will also be close to each other in the 3D structure of chromatin.

Analysis of lineage-specific Alu subfamilies in the genome of the olive baboon, Papio anubis

BackgroundAlu elements are primate-specific retroposons that mobilize using the enzymatic machinery of L1 s. The recently completed baboon genome project found that the mobilization rate of Alu elements is higher than in the genome of any other primate studied thus far. However, the Alu subfamily structure present in and specific to baboons had not been examined yet.ResultsHere we report 129 Alu subfamilies that are propagating in the genome of the olive baboon, with 127 of these subfamilies being new and specific to the baboon lineage. We analyzed 233 Alu insertions in the genome of the olive baboon using locus specific polymerase chain reaction assays, covering 113 of the 129 subfamilies. The allele frequency data from these insertions show that none of the nine groups of subfamilies are nearing fixation in the lineage.ConclusionsMany subfamilies of Alu elements are actively mobilizing throughout the baboon lineage, with most being specific to the baboon lineage.

Recently integrated Alu insertions in the squirrel monkey (Saimiri) lineage and application for population analyses

BackgroundThe evolution of Alu elements has been ongoing in primate lineages and Alu insertion polymorphisms are widely used in phylogenetic and population genetics studies. Alu subfamilies in the squirrel monkey (Saimiri), a New World Monkey (NWM), were recently reported. Squirrel monkeys are commonly used in biomedical research and often require species identification. The purpose of this study was two-fold: 1) Perform locus-specific PCR analyses on recently integrated Alu insertions in Saimiri to determine their amplification dynamics, and 2) Identify a subset of Alu insertion polymorphisms with species informative allele frequency distributions between the Saimiri sciureus and Saimiri boliviensis groups.ResultsPCR analyses were performed on a DNA panel of 32 squirrel monkey individuals for 382 Alu insertion events ≤2% diverged from 46 different Alu subfamily consensus sequences, 25 Saimiri specific and 21 NWM specific Alu subfamilies. Of the 382 loci, 110 were polymorphic for presence / absence among squirrel monkey individuals, 35 elements from 14 different Saimiri specific Alu subfamilies and 75 elements from 19 different NWM specific Alu subfamilies (13 of 46 subfamilies analyzed did not contain polymorphic insertions). Of the 110 Alu insertion polymorphisms, 51 had species informative allele frequency distributions between Saimiri sciureus and Saimiri boliviensis groups.ConclusionsThis study confirms the evolution of Alu subfamilies in Saimiri and provides evidence for an ongoing and prolific expansion of these elements in Saimiri with many active subfamilies concurrently propagating. The subset of polymorphic Alu insertions with species informative allele frequency distribution between Saimiri sciureus and Saimiri boliviensis will be instructive for specimen identification and conservation biology.

Evolution of Alu Subfamily Structure in the Saimiri Lineage of New World Monkeys.

Squirrel monkeys, Saimiri, are commonly found in zoological parks and used in biomedical research. S. boliviensis is the most common species for research; however, there is little information about genome evolution within this primate lineage. Here, we reconstruct the Alu element sequence amplification and evolution in the genus Saimiri at the time of divergence within the family Cebidae lineage. Alu elements are the most successful SINE (Short Interspersed Element) in primates. Here, we report 46 Saimiri lineage specific Alu subfamilies. Retrotransposition activity involved subfamilies related to AluS, AluTa10, and AluTa15. Many subfamilies are simultaneously active within the Saimiri lineage, a finding which supports the stealth model of Alu amplification. We also report a high resolution analysis of Alu subfamilies within the S. boliviensis genome [saiBol1].

Alu SINE analyses of 3,000-year-old human skeletal remains: a pilot study.

BackgroundAs Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter.ResultsFor 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp.ConclusionsOur data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0063-y) contains supplementary material, which is available to authorized users.

DNA methylation variation of human-specific Alu repeats

ABSTRACTDNA methylation is the major repression mechanism for human retrotransposons, such as the Alu family. Here, we have determined the methylation levels associated with 5238 loci belonging to 2 Alu subfamilies, AluYa5 and AluYb8, using high-throughput targeted repeat element bisulfite sequencing (HT-TREBS). The results indicate that ∼90% of loci are repressed by high methylation levels. Of the remaining loci, many of the hypomethylated elements are found near gene promoters and show high levels of DNA methylation variation. We have characterized this variation in the context of tumorigenesis and interindividual differences. Comparison of a primary breast tumor and its matched normal tissue revealed early DNA methylation changes in ∼1% of AluYb8 elements in response to tumorigenesis. Simultaneously, AluYa5/Yb8 elements proximal to promoters also showed differences in methylation of up to one order of magnitude, even between normal individuals. Overall, the current study demonstrates that early loss of methylation occurs during tumorigenesis in a subset of young Alu elements, suggesting their potential clinical relevance. However, approaches such as deep-bisulfite-sequencing of individual loci using HT-TREBS are required to distinguish clinically relevant loci from the background observed for AluYa5/Yb8 elements in general with regard to high levels of interindividual variation in DNA methylation.

The domain structure and distribution of Alu elements in long noncoding RNAs and mRNAs.

Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3′ and 5′ ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions.

Discovery and characterization ofAlurepeat sequences via precise local read assembly

Alu insertions have contributed to >11% of the human genome and ∼30–35 Alu subfamilies remain actively mobile, yet the characterization of polymorphic Alu insertions from short-read data remains a challenge. We build on existing computational methods to combine Alu detection and de novo assembly of WGS data as a means to reconstruct the full sequence of insertion events from Illumina paired end reads. Comparison with published calls obtained using PacBio long-reads indicates a false discovery rate below 5%, at the cost of reduced sensitivity due to the colocation of reference and non-reference repeats. We generate a highly accurate call set of 1614 completely assembled Alu variants from 53 samples from the Human Genome Diversity Project (HGDP) panel. We utilize the reconstructed alternative insertion haplotypes to genotype 1010 fully assembled insertions, obtaining >99% agreement with genotypes obtained by PCR. In our assembled sequences, we find evidence of premature insertion mechanisms and observe 5′ truncation in 16% of AluYa5 and AluYb8 insertions. The sites of truncation coincide with stem-loop structures and SRP9/14 binding sites in the Alu RNA, implicating L1 ORF2p pausing in the generation of 5′ truncations. Additionally, we identified variable AluJ and AluS elements that likely arose due to non-retrotransposition mechanisms.

The expression of tRNA genes and young Alu subfamilies in human tumor cells U937 during apoptosis

Expression of RNA polymerase III-directed genes, tRNAimet1, tRNAHis, and Alu-repeats belonging to the youngest subfamilies AluY (AluYa5 and AluYb8), have been studied in human U937 cells during camptothecin-induced apoptosis. The level of tRNAiMet1 increased 1.5-fold, the tRNAHis level did not change, and the level of AluY-RNA increased fourto ten-fold after 6 h of camptothecin (CAM) treatment compared to control (nonapoptotic) cells. We also studied the level of AluYb8 DNA methylation in apoptotic U937 cells. We have shown that the level of AluYb8 DNA methylation does not change at different stages of apoptosis and does not differ in apoptotic and control cells. We assume that the increase in gene expression of young AluY repeats and tRNAiMet1 is critical for realization of the apoptotic pathways in cells.

Sequence Analysis and Characterization of Active Human Alu Subfamilies Based on the 1000 Genomes Pilot Project.

The goal of the 1000 Genomes Consortium is to characterize human genome structural variation (SV), including forms of copy number variations such as deletions, duplications, and insertions. Mobile element insertions, particularly Alu elements, are major contributors to genomic SV among humans. During the pilot phase of the project we experimentally validated 645 (611 intergenic and 34 exon targeted) polymorphic “young” Alu insertion events, absent from the human reference genome. Here, we report high resolution sequencing of 343 (322 unique) recent Alu insertion events, along with their respective target site duplications, precise genomic breakpoint coordinates, subfamily assignment, percent divergence, and estimated A-rich tail lengths. All the sequenced Alu loci were derived from the AluY lineage with no evidence of retrotransposition activity involving older Alu families (e.g., AluJ and AluS). AluYa5 is currently the most active Alu subfamily in the human lineage, followed by AluYb8, and many others including three newly identified subfamilies we have termed AluYb7a3, AluYb8b1, and AluYa4a1. This report provides the structural details of 322 unique Alu variants from individual human genomes collectively adding about 100 kb of genomic variation. Many Alu subfamilies are currently active in human populations, including a surprising level of AluY retrotransposition. Human Alu subfamilies exhibit continuous evolution with potential drivers sprouting new Alu lineages.

Alu expression profiles as a novel RNA signature in biology and disease

SINE retrotransposons of the Alu subfamily are the most numerous active mobile DNA elements in the human genome. Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, but activated in response to viral infection, genotoxic anticancer agents and other stimuli, through uncharacterized epigenetic switches interspersed throughout the genome. The elucidation of Alu RNA roles in cell biology and pathology has long been hampered by difficulties in their profiling at single-locus resolution, due to their repetitive nature. We recently found how to overcome this limitation by computational screening of RNA-seq data, thus opening the way to Alu transcriptome profiling as a novel tool to explore disease-related epigenome alterations.