Changes in Gene Expression and DNA Methylation of Evolutionarily Young AluY Repeats during Apoptosis of Human K562 Erythro-Myeloblastic Leukemia Cells

Using a human K562 erythromyeloblastoid cell culture, we demonstrated changes in gene expression of Alu repeats, members of evolutionarily young AluY subfamilies (human mobile SINE elements), and in the DNA methylation level of AluYb8 during camptothecin (CAM)-induced apoptosis. The AluY-RNA level increased about 10 times 24 h and 20 times 48 h after exposure to CAM vs. proliferating cells. Using methylation-specific (MSP) PCR and high-resolution melting (HRM), we showed that the overall AluYb8-DNA methylation level remained intact throughout the apoptotic stages. Using DNA sequencing after bisulfite conversion, we established that at the CpG site, located in the A'-box of the AluYb8 gene promoter, the methylation level decreased significantly during different apoptotic stages. Apparently, it is reduced CpG methylation at the A'-box of the AluYb8 gene promoter, discovered in this work, that is one of the possible factors which account for increased expression of AluY repeats during K562 cell apoptosis. We assume that increased gene expression of evolutionarily young AluY repeats plays an important role in the implementation of the cellular apoptotic pathway.     

DNA methylation variation of human-specific Alu repeats

DNA 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.     

Polymorphic Alu insertions within the Major Histocompatibility Complex class I genomic region: a brief review

Most polymorphic Alu insertions (POALINs) belong to a subgroup of the Alu multicopy retrotransposon family of short interspersed nucleotide elements (SINEs) that are categorized as AluYb8 and AluYa5. The number of AluYb8/AluYa5 members (approximately 4,492 copies) is significantly less than the approximately one million fixed Alu copies per human genome. We have studied the presence of POALINs within the Major Histocompatibility Complex (MHC) class I region on the short arm of chromosome 6 (6p21.3) because this region has a high gene density, many genes with immune system functions, large sequence variations and diversity, duplications and redundancy, and a strong association with more than 100 different diseases. Since little is known about POALINs within the MHC genomic region, we undertook to identify some of the members of the AluYb8/AluYa5 subfamily and to study their frequency of distribution and genetic characteristics in different populations. As a result of our comparative genomic analyses, we identified the insertion sites for five POALINs distributed within the MHC class I region. This brief review outlines the locations of the insertions and sequence features of the five MHC POALINs, their single site and haplotype frequencies in different geographic populations, and their association with different HLA class I genes and disease. We show that the MHC POALINs have a potential value as lineage and linkage markers for the study of human population genetics, disease associations, genomic diversity and evolution.     

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.     

Evolution of an Alu DNA element of type Sx in the lineage of primates and the origin of an associated tetranucleotide microsatellite.

A 394-bp DNA fragment, which in human is on chromosome 6 near the MOG (myelin oligodendrocyte glycoprotein) gene and encompasses an Alu element and an associated tetranucleotide microsatellite, was sequenced from a large range of primate species to follow its evolutionary divergence and to understand the origin of the microsatellite. This Alu element is found at the same orthologous position in all primates sequenced, but the tetranucleotide repeat is present only in Catarrhini between the 3'-oligo(dA) of the Alu element and the 3' flanking direct repeat. Little intraspecific variation was found. Sequence identity values for this orthologous primate Alu averaged 90% (82-99%) with transitions comprising between 70% and 100% of the observed nucleotide substitutions. Although the insertion of the Alu element predates the separation of these species, the original sequence of the site of integration can still be identified. This identification of the direct repeats suggests an active role of the oligo(dA) of the Alu element in the origin of the tetranucleotide repeats. The microsatellite probably appeared after the insertion of the Alu element, early in the lineage leading to the common ancestor of the hominoids and the Old World monkeys.     

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.     

Molecular evolution of intergenic DNA in higher primates: pattern of DNA changes, molecular clock, and evolution of repetitive sequences

A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.      

Evolution of alu family repeats since the divergence of human and chimpanzee

Summary The DNA sequences of three members of the Alu family of repeated sequences located 5 to the chimpanzee 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human.     

DNA sequence polymorphisms in Alu repeats

We have developed an efficient method for detection of sequence differences in genomic DNA based on a new principle (M. Orita et al., 1989, Genomics 5: 874-879). Using this method, we show here that approximately half the Alu repeats interspersed in the human genome are significantly polymorphic. Analysis of Alu repeat polymorphism should be useful in construction of a high-resolution map and also in identifying genotypes of individuals for clinical and other purposes because the repeats are ubiquitous and the technique for their detection is simple.     

Hidden ancient repeats in DNA: Mapping and quantification

We have shown, in a previous paper, that tandem repeating sequences, especially triplet repeats, play a very important role in gene evolution. This result led to the formulation of the following hypothesis: most of the genomic sequences evolved through everlasting acts of tandem repeat expansions with subsequent accumulation of changes. In order to estimate how much of the observed sequences have the repeat origin we describe the adaptation of a text segmentation algorithm, based on dynamic programming, to the mapping of the ancient expansion events. The algorithm maximizes the segmentation cost, calculated as the similarity of obtained fragments to the putative repeat sequence. In the first application of the algorithm to segmentations of genomic sequences, a significant difference between the natural sequences and the corresponding shuffled sequences is detected. The natural fragments are longer and more similar to the putative repeat sequences. As our analysis shows, the coding sequences allow for repeats only when the size of the repeated words is divisible by three. In contrast, in the non-coding sequences, all repeated word sizes are present. It was estimated, that in Escherichia coli K12 genome, about 35.5% of sequence can be detectably traced to original simple repeat ancestors. The results shed light on the genomic sequence organization, and strongly confirm the hypothesis about the crucial role of triplet expansions in gene origin and evolution.     

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.     

Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.     

Single replication origin of the archaeon Methanosarcina mazei revealed by the Z curve method

The genomic sequence of the archaeon Methanosarcina mazei has been analyzed by the Z curve method. The Z curve is a three-dimensional curve that uniquely represents the given DNA sequence. The three-dimensional Z curve and its x and y components for the genome of M. mazei show a sharp peak and relatively broad peak, respectively. The cdc6 gene is located exactly at the position of the sharp peak. Based on the known behavior of the Z curves for the archaea whose replication origins have been identified, we hypothesize that the replication origin and termination sites correspond to the positions of the sharp peak and broad peak, respectively. We have located an intergenic region that is between the cdc6 gene (MM1314) and the gene for an adjacent protein (MM1315), which shows strong characteristics of the known replication origins. This region is highly rich in AT and contains multiple copies of consecutive repeats. Our results strongly suggest that the single replication origin of M. mazei is situated at the intergenic region between the cdc6 gene and the gene for the adjacent protein, from 1,564,657 to 1,566,241 bp of the genome.     

HIGM syndrome caused by insertion of an AluYb8 element in exon 1 of the <Emphasis Type="Italic">CD40LG</Emphasis> gene

A new mutation of the CD40LG gene that encodes the CD40 ligand molecule was characterized in a young patient harboring a hyper-IgM with immunodeficiency syndrome. Inactivation of CD40LG gene resulted from the insertion of an AluYb8 element in exon 1 responsible for a total deficiency of CD40 ligand expression by T lymphocytes. Maternal transmission of the X-linked mutation was confirmed by gene-specific polymerase chain reaction. This is the 17th case report concerning a human genetic disease caused by an Alu element insertion in a coding sequence.     

Origin and instability of GAA repeats: insights from Alu elements

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7- 38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3' end of an "A" stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed.     

The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.     

Junctions between genes in the haptoglobin gene cluster of primates.

To investigate the nature of the recombination that generated the haptoglobin three-gene cluster in Old World primates, we sequenced the region between the second gene (HPR) and the third gene (HPP) in chimpanzees (15 kb), as well as the region 3' to the cluster in humans (14 kb). Comparison to the previously sequenced human haptoglobin (HP) and HPR genes showed that the junction point between HP and HPR in humans (junction 1) was not identical to the junction point between the HPR and HPP genes of the chimpanzee (junction 2). An Alu sequence was found at each junction, but both Alu sequences lacked short direct repeats of the flanking genomic DNA. The lack of direct repeats implies that both junction Alu sequences are the products of recombination between different Alu elements. In addition, other insertion and deletion events are clustered in the regions near the junction Alu sequences. The observation that Alu sequences define the junctions between genes in the haptoglobin gene cluster emphasizes the importance of Alu sequences in the evolution of multigene families.