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.     

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.     

Large-scale analysis of the Alu Ya5 and Yb8 subfamilies and their contribution to human genomic diversity

We have utilized computational biology to screen GenBank for the presence of recently integrated Ya5 and Yb8 Alu family members. Our analysis identified 2640 Ya5 Alu family members and 1852 Yb8 Alu family members from the draft sequence of the human genome. We selected a set of 475 of these elements for detailed analyses. Analysis of the DNA sequences from the individual Alu elements revealed a low level of random mutations within both subfamilies consistent with the recent origin of these elements within the human genome. Polymerase chain reaction assays were used to determine the phylogenetic distribution and human genomic variation associated with each Alu repeat. Over 99 % of the Ya5 and Yb8 Alu family members were restricted to the human genome and absent from orthologous positions within the genomes of several non-human primates, confirming the recent origin of these Alu subfamilies in the human genome. Approximately 1 % of the analyzed Ya5 and Yb8 Alu family members had integrated into previously undefined repeated regions of the human genome. Analysis of mosaic Yb8 elements suggests gene conversion played an important role in generating sequence diversity among these elements. Of the 475 evaluated elements, a total of 106 of the Ya5 and Yb8 Alu family members were polymorphic for insertion presence/absence within the genomes of a diverse array of human populations. The newly identified Alu insertion polymorphisms will be useful tools for the study of human genomic diversity.     

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.     

Recently integrated human Alu repeats: finding needles in the haystack

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

LINE-1 preTa elements in the human genome

The preTa subfamily of long interspersed elements (LINEs) is characterized by a three base-pair "ACG" sequence in the 3' untranslated region, contains approximately 400 members in the human genome, and has low level of nucleotide divergence with an estimated average age of 2.34 million years old suggesting that expansion of the L1 preTa subfamily occurred just after the divergence of humans and African apes. We have identified 362 preTa L1 elements from the draft human genomic sequence, investigated the genomic characteristics of preTa L1 insertions, and screened individual elements across diverse human populations and various non-human primate species using polymerase chain reaction (PCR) assays to determine the phylogenetic origin and levels of human genomic diversity associated with the L1 elements. All of the preTa L1 elements analyzed by PCR were absent from the orthologous positions in non-human primate genomes with 33 (14%) of the L1 elements being polymorphic with respect to insertion presence or absence in the human genome. The newly identified L1 insertion polymorphisms will prove useful as identical by descent genetic markers for the study of human population genetics. We provide evidence that preTa L1 elements show an integration site preference for genomic regions with low GC content. Computational analysis of the preTa L1 elements revealed that 29% of the elements amenable to complete sequence analysis have apparently escaped 5' truncation and are essentially full-length (approximately 6kb). In all, 29 have two intact open reading frames and may be capable of retrotransposition.     

Non-traditional Alu evolution and primate genomic diversity

Alu elements belonging to the previously identified "young" subfamilies are thought to have inserted in the human genome after the divergence of humans from non-human primates and therefore should not be present in non-human primate genomes. Polymerase chain reaction (PCR) based screening of over 500 Alu insertion loci resulted in the recovery of a few "young" Alu elements that also resided at orthologous positions in non-human primate genomes. Sequence analysis demonstrated these "young" Alu insertions represented gene conversion events of pre-existing ancient Alu elements or independent parallel insertions of older Alu elements in the same genomic region. The level of gene conversion between Alu elements suggests that it may have a significant influence on the single nucleotide diversity within the genome. All the instances of multiple independent Alu insertions within the same small genomic regions were recovered from the owl monkey genome, indicating a higher Alu amplification rate in owl monkeys relative to many other primates. This study suggests that the majority of Alu insertions in primate genomes are the products of unique evolutionary events.     

A human-specific subfamily of Alu sequences.

Of a total of 500,000 Alu family members, approximately 500 are present as a human-specific (HS) subfamily. Each of the HS subfamily members shares a high degree of nucleotide identity and is not present at orthologous positions in other primate genomes, suggesting that HS subfamily members have recently inserted within the human genome. This confirms the hypothesis that the majority of Alu family members are amplified copies of a "master" gene(s). This master gene appears to be amplifying at a rate much slower than that seen earlier in primate evolution. Some of the HS Alu subfamily members have amplified so recently that they are dimorphic in the human population, making them a potentially powerful tool for studies of human populations.     

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.     

Identification and characterization of two polymorphic Ya5 Alu repeats

Two new polymorphic Alu elements (HS2.25 and HS4.14) belonging to the young (Ya5/8) subfamily of human-specific Alu repeats have been identified. DNA sequence analysis of both Alu repeats revealed that each Alu repeat had a long 3′-oligo-dA-rich tail (41 and 52 nucleotides in length) and a low level of random mutations. HS2.25 and HS4.14 were flanked by short precise direct repeats of 8 and 14 nucleotides in length, respectively. HS2.25 was located on human chromosome 13, and HS4.14 on chromosome 1. Both Alu elements were absent from the orthologous positions within the genomes of non-human primates, and were highly polymorphic in a survey of twelve geographically diverse human groups.     

A comprehensive analysis of recently integrated human Ta L1 elements

The Ta (transcribed, subset a) subfamily of L1 LINEs (long interspersed elements) is characterized by a 3-bp ACA sequence in the 3' untranslated region and contains approximately 520 members in the human genome. Here, we have extracted 468 Ta L1Hs (L1 human specific) elements from the draft human genomic sequence and screened individual elements using polymerase-chain-reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. One hundred twenty-four of the elements amenable to complete sequence analysis were full length ( approximately 6 kb) and have apparently escaped any 5' truncation. Forty-four of these full-length elements have two intact open reading frames and may be capable of retrotransposition. Sequence analysis of the Ta L1 elements showed a low level of nucleotide divergence with an estimated age of 1.99 million years, suggesting that expansion of the L1 Ta subfamily occurred after the divergence of humans and African apes. A total of 262 Ta L1 elements were screened with PCR-based assays to determine their phylogenetic origin and the level of human genomic variation associated with each element. All of the Ta L1 elements analyzed by PCR were absent from the orthologous positions in nonhuman primate genomes, except for a single element (L1HS72) that was also present in the common (Pan troglodytes) and pygmy (P. paniscus) chimpanzee genomes. Sequence analysis revealed that this single exception is the product of a gene conversion event involving an older preexisting L1 element. One hundred fifteen (45%) of the Ta L1 elements were polymorphic with respect to insertion presence or absence and will serve as identical-by-descent markers for the study of human evolution.     

A recent insertion of an alu element on the Y chromosome is a useful marker for human population studies

A member of the Alu family of repeated DNA elements has been identified on the long arm of the human Y chromosome, Yq11. This element, referred to as the Y Alu polymorphic (YAP) element, is present at a specific site on the Y chromosome in some humans and is absent in others. Phylogenetic comparisons with other Alu sequences reveal that the YAP element is a member of the polymorphic subfamily-3 (PSF-3), a previously undefined subfamily of Alu elements. The evolutionary relationships of PSF-3 to other Alu subfamilies support the hypothesis that recently inserted elements result from multiple source genes. The frequency of the YAP element is described in 340 individuals from 14 populations, and the data are combined with those from other populations. There is both significant heterogeneity among populations and a clear pattern in the frequencies of the insertion: sub-Saharan Africans have the highest frequencies, followed by northern Africans, Europeans, Oceanians, and Asians. An interesting exception is the relatively high frequency of the YAP element in Japanese. The greatest genetic distance is observed between the African and non-African populations. The YAP is especially useful for studying human population history from the perspective of male lineages.      

In search of polymorphic Alu insertions with restricted geographic distributions

Alu elements are transposable elements that have reached over one million copies in the human genome. Some Alu elements inserted in the genome so recently that they are still polymorphic for insertion presence or absence in human populations. Recently, there has been an increasing interest in using Alu variation for studies of human population genetic structure and inference of individual geographic origin. Currently, this requires a high number of Alu loci. Here, we used a linker-mediated polymerase chain reaction method to preferentially identify low-frequency Alu elements in various human DNA samples with different geographic origins. The candidate Alu loci were subsequently genotyped in 18 worldwide human populations (approximately 370 individuals), resulting in the identification of two new Alu insertions restricted to populations of African ancestry. Our results suggest that it may ultimately become possible to correctly infer the geographic affiliation of unknown samples with high levels of confidence without having to genotype as many as 100 Alu loci. This is desirable if Alu insertion polymorphisms are to be used for human evolution studies or forensic applications.     

PROGINS Alu insertion and human genomic diversity

A polymorphic Alu element belonging to the young Ya5 subfamily of Alu repeats located in the progesterone receptor gene has been characterized. Using a polymerase chain reaction (PCR)-based assay, the genetic diversity associated with the PROGINS Alu repeat was determined in a diverse array of human populations. The level of insertion polymorphism associated with PROGINS suggests that it will be a useful marker for the study of human evolution. In addition, we determined the distribution of the PROGINS Alu insertion in two groups of women from greater New Orleans, LA with breast cancer. The PROGINS Alu insertion was not associated with breast cancer in the populations tested.     

Mono-allelic retrotransposon insertion addresses epigenetic transcriptional repression in human genome

Background

Retrotransposons have been extensively studied in plants and animals and have been shown to have an impact on human genome dynamics and evolution. Their ability to move within genomes gives retrotransposons to affect genome instability.

Methods

we examined the polymorphic inserted AluYa5, evolutionary young Alu, in the progesterone receptor gene to determine the effects of Alu insertion on molecular environment. We used mono-allelic inserted cell lines which carry both Alu-present and Alu-absent alleles. To determine the epigenetic change and gene expression, we performed restriction enzyme digestion, Pyrosequencing, and Chromatin Immunoprecipitation.

Results

We observed that the polymorphic insertion of evolutionally young Alu causes increasing levels of DNA methylation in the surrounding genomic area and generates inactive histone tail modifications. Consequently the Alu insertion deleteriously inactivates the neighboring gene expression.

Conclusion

The mono-allelic Alu insertion cell line clearly showed that polymorphic inserted repetitive elements cause the inactivation of neighboring gene expression, bringing aberrant epigenetic changes.     

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.