The Role of Recombination in the Origin and Evolution of Alu Subfamilies

Alus are the most abundant and successful short interspersed nuclear elements found in primate genomes. In humans, they represent about 10% of the genome, although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. In this study, we have addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination, which stimulates further research regarding the potential of chimeric active Alus to punctuate the genome.     

Comprehensive analysis of two Alu Yd subfamilies

Alu elements have inserted in the human genome throughout primate evolution. A small number of Alu insertions have occurred after the divergence of humans from nonhuman primates and therefore should not be present in nonhuman primate genomes. Most of these recently integrated Alu elements are contained with a series of discrete Alu subfamilies that are related to each other based upon diagnostic nucleotide substitutions. We have extracted members of the Alu Yd subfamily that are derivatives of the Alu Y subfamily that share a common 12-bp deletion that defines the Yd lineage from the draft sequence of the human genome. Analysis of the Yd Alu elements resulted in the recovery of two new Alu subfamilies, Yd3 and Yd6, which contain a total of 295 members (198 Yd3 and 97 Yd6). DNA sequence analysis of each of the Alu Yd subfamilies yielded age estimates of 8.02 and 1.20 million years old for the Alu Yd3 and Yd6 subfamilies, respectively. Two hundred Alu Yd3 and Yd6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and associated levels of human genomic diversity. The Alu Yd3 subfamily appears to have started amplifying relatively early in primate evolution and continued propagating albeit at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Only two of the elements are polymorphic in the human genome and absent from the genomes of nonhuman primates. By contrast all of the members of the Alu Yd6 subfamily are restricted to the human genome, with 12% of the elements representing insertion polymorphisms in human populations. A single Alu Yd6 locus contained an independent parallel forward insertion of a paralogous Alu Sq sequence in the owl monkey. These Alu subfamilies are a source of genomic fossil relics for the study of primate phylogenetics and human population genetics.     

Most recent AluY insertions in human gene introns reduce the content of the primary transcripts in a cell type specific manner

Being the most effectively transposed primate-specific SINEs, Alu elements are present in more than one million copies in the human genome and include most recently transposed subsets of AluY elements that are polymorphic in humans. Although Alu elements are commonly thought to play an essential role in shaping and functioning of primate genomes, the understanding of the impact of recent Alu insertions on human gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for AluY insertions in their introns in human cell lines of various origins. We demonstrated that some AluY insertions correlated with decreased content of the corresponding hnRNAs. The effect observed does not depend on sequences of Alu elements and their orientation but is likely to be cell type specific.     

Alu element mutation spectra: molecular clocks and the effect of DNA methylation

In primate genomes more than 40% of CpG islands are found within repetitive elements. With more than one million copies in the human genome, the Alu family of retrotransposons represents the most successful short interspersed element (SINE) in primates and CpG dinucleotides make up about 20% of Alu sequences. It is generally thought that CpG dinucleotides mutate approximately ten times faster than other dinucleotides due to cytosine methylation and the subsequent deamination and conversion of C-->T. However, the disparity of Alu subfamily age estimations based upon CpG or non-CpG substitution density indicates a more complex relationship between CpG and non-CpG substitutions within the Alu elements. Here we report an analysis of the mutation patterns for 5296 Alu elements comprising 20 subfamilies. Our results indicate a relatively constant CpG versus non-CpG substitution ratio of approximately 6 for the young (AluY) and intermediate (AluS) Alu subfamilies. However, a more complex non-linear relationship between CpG and non-CpG substitutions was observed when old (AluJ) subfamilies were included in the analysis. These patterns may be the result of the slowdown of the neutral mutation rate during primate evolution and/or an increase in the CpG mutation rate as the consequence of increased DNA methylation in response to a burst of retrotransposition activity approximately 35 million years ago.     

An Alu transposition model for the origin and expansion of human segmental duplications

Relative to genomes of other sequenced organisms, the human genome appears particularly enriched for large, highly homologous segmental duplications (> or =90% sequence identity and > or =10 kbp in length). The molecular basis for this enrichment is unknown. We sought to gain insight into the mechanism of origin, by systematically examining sequence features at the junctions of duplications. We analyzed 9,464 junctions within regions of high-quality finished sequence from a genomewide set of 2,366 duplication alignments. We observed a highly significant (P<.0001) enrichment of Alu short interspersed element (SINE) sequences near or within the junction. Twenty-seven percent of all segmental duplications terminated within an Alu repeat. The Alu junction enrichment was most pronounced for interspersed segmental duplications separated by > or =1 Mb of intervening sequence. Alu elements at the junctions showed higher levels of divergence, consistent with Alu-Alu-mediated recombination events. When we classified Alu elements into major subfamilies, younger elements (AluY and AluS) accounted for the enrichment, whereas the oldest primate family (AluJ) showed no enrichment. We propose that the primate-specific burst of Alu retroposition activity (which occurred 35-40 million years ago) sensitized the ancestral human genome for Alu-Alu-mediated recombination events, which, in turn, initiated the expansion of gene-rich segmental duplications and their subsequent role in nonallelic homologous recombination.     

Epigenetic control of retrotransposon expression in human embryonic stem cells

Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.     

Analysis of the human Alu Ya-lineage

The Alu Ya-lineage is a group of related, short interspersed elements (SINEs) found in primates. This lineage includes subfamilies Ya1-Ya5, Ya5a2 and others. Some of these subfamilies are still actively mobilizing in the human genome. We have analyzed 2482 elements that reside in the human genome draft sequence and focused our analyses on the 2318 human autosomal Ya Alu elements. A total of 1470 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual Ya-lineage Alu elements. About 22% (313/1452) of the Ya-lineage Alu elements were polymorphic for the insertion presence on human autosomes. Less than 0.01% (5/1452) of the Ya-lineage loci analyzed displayed insertions in orthologous loci in non-human primate genomes. DNA sequence analysis of the orthologous inserts showed that the orthologous loci contained older pre-existing Y, Sc or Sq Alu subfamily elements that were the result of parallel forward insertions or involved in gene conversion events in the human lineage. This study is the largest analysis of a group of "young", evolutionarily related human subfamilies. The size, evolutionary age and variable allele insertion frequencies of several of these subfamilies makes members of the Ya-lineage useful tools for human population studies and primate phylogenetics.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

Recently integrated Alu elements and human genomic diversity

A comprehensive analysis of two Alu Y lineage subfamilies was undertaken to assess Alu-associated genomic diversity and identify new Alu insertion polymorphisms for the study of human population genetics. Recently integrated Alu elements (283) from the Yg6 and Yi6 subfamilies were analyzed by polymerase chain reaction (PCR), and 25 of the loci analyzed were polymorphic for insertion presence/absence within the genomes of a diverse array of human populations. These newly identified Alu insertion polymorphisms will be useful tools for the study of human genomic diversity. Our screening of the Alu insertion loci also resulted in the recovery of several "young" Alu elements that resided at orthologous positions in nonhuman primate genomes. Sequence analysis demonstrated these "young" Alu insertions were the products of gene conversion events of older, preexisting Alu elements or independent parallel forward insertions of older Alu elements in the same short genomic region. The level of gene conversion between Alu elements suggests that it may have an influence on the single nucleotide polymorphism within Alu elements in the genome. We have also identified two genomic deletions associated with the retroposition and insertion of Alu Y lineage elements into the human genome. This type of Alu retroposition-mediated genomic deletion is a novel source of lineage-specific evolution within primate genomes.     

The Alu Yc1 subfamily: sorting the wheat from the chaff

Members of the Alu Yc1 subfamily are distinguished from the older Alu Y subfamily by a signature G-->A substitution at base 148 of their 281-bp consensus sequence. Members of the much older and larger Alu Y subfamily could have by chance accumulated this signature G-->A substitution and be misclassified as belonging to the Alu Yc1 subfamily. Using a Mahanalobis classification method, it was estimated that the "authentic" Alu Yc1 subfamily consists of approximately 262 members in the human genome. PCR amplification and further analysis was successfully completed on 225 of the Yc1 Alu family members. One hundred and seventy-seven Yc1 Alu elements were determined to be monomorphic (fixed for presence) in a panel of diverse human genomes. Forty-eight of the Yc1 Alu elements were polymorphic for insertion presence/absence in diverse human genomes. The insertion polymorphism rate of 21% in the human genome is similar to rates reported previously for other "young" Alu subfamilies. The polymorphic Yc1 Alu elements will be useful genetic loci for the study of human population genetics.     

Comprehensive analysis of Alu-associated diversity on the human sex chromosomes

A comprehensive analysis of the human sex chromosomes was undertaken to assess Alu-associated human genomic diversity and to identify novel Alu insertion polymorphisms for the study of human evolution. Three hundred forty-five recently integrated Alu elements from eight different Alu subfamilies were identified on the X and Y chromosomes, 225 of which were selected and analyzed by polymerase chain reaction (PCR). From a total of 225 elements analyzed, 16 were found to be polymorphic on the X chromosome and one on the Y chromosome. In line with previous research using other classes of genetic markers, our results indicate reduced Alu-associated insertion polymorphism on the human sex chromosomes, presumably reflective of the reduced recombination rates and lower effective population sizes on the sex chromosomes. The Alu insertion polymorphisms identified in this study should prove useful for the study of human population genetics.     

Which transposable elements are active in the human genome?

Although a large proportion (44%) of the human genome is occupied by transposons and transposon-like repetitive elements, only a small proportion (<0.05%) of these elements remain active today. Recent evidence indicates that approximately 35-40 subfamilies of Alu, L1 and SVA elements (and possibly HERV-K elements) remain actively mobile in the human genome. These active transposons are of great interest because they continue to produce genetic diversity in human populations and also cause human diseases by integrating into genes. In this review, we examine these active human transposons and explore mechanistic factors that influence their mobilization.     

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 ± 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 ± 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.     

Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution.

The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolution. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.     

An unusual Alu repeat sequence within the CAD gene

Summary There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangments may be a factor in generating the diversity of Alu repeats found in primate genomes.