The Distribution of L1 and Alu Retroelements in Relation to GC Content on Human Sex Chromosomes Is Consistent with the Ectopic Recombination Model

The distribution of Alu and L1 retroelements in the human genome changes with their age. Active retroelements target AT-rich regions, but their frequency increases in GC- and gene-rich regions of the genome with increasing age of the insertions. Currently there is no consensus on the mechanism generating this pattern. In this paper we test the hypothesis that selection against deleterious deletions caused by ectopic recombination between repeats is the main cause of the inhomogeneous distribution of L1s and Alus, by means of a detailed analysis of the GC distribution of the repeats on the sex chromosomes. We show that (1) unlike on the autosomes and X chromosome, L1s do not accumulate on the Y chromosome in GC-rich regions, whereas Alus accumulate there to a minor extent; (2) on the Y chromosome Alu and L1 densities are positively correlated, unlike the negative correlation on other chromosomes; and (3) in gene-poor regions of chromosome 4 and X, the distribution of Alus and L1s does not shift toward GC-rich regions. In addition, we show that although local GC content of long L1 insertions is lower than average, their selective loss from recombining chromosomes is not the main cause of the enrichment of ancient L1s in GC-rich regions. The results support the hypothesis that ectopic recombination causes the shift of Alu and L1 distributions toward the gene-rich regions of the genome. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. Deborah Charlesworth     

Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.     

Clustering of Genes Coding for DNA Binding Proteins in a Regionof Atypical Evolution of the Human Genome

Comparison of the human and mouse genomes has revealed that significant variations in evolutionary rates exist among genomic regions and that a large part of this variation is interchromosomal. We confirm in this work, using a large collection of introns, that human chromosome 19 is the one that shows the highest divergence with respect to mouse. To search for other differences among chromosomes, we examine the distribution of gene functions in human and mouse chromosomes using the Gene Ontology definitions. We found by correspondence analysis that among the strongest clusterings of gene functions in human chromosomes is a group of genes coding for DNA binding proteins in chromosome 19. Interestingly, chromosome 19 also has a very high GC content, a feature that has been proposed to promote an opening of the chromatin, thereby facilitating binding of proteins to the DNA helix. In the mouse genome, however, a similar aggregation of genes coding for DNA binding proteins and high GC content cannot be found. This suggests that the distribution of genes coding for DNA binding proteins and the variations of the chromatin accessibility to these proteins are different in the human and mouse genomes. It is likely that the overall high synonymous and intron rates in chromosome 19 are a by-product of the high GC content of this chromosome.Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18, 08034 Barcelona, Spain     

The Biased Distribution of Alus in Human Isochores Might Be Driven by Recombination

Alu retrotransposons do not show a homogeneous distribution over the human genome but have a higher density in GC-rich (H) than in AT-rich (L) isochores. However, since they preferentially insert into the L isochores, the question arises: What is the evolutionary mechanism that shifts the Alu density maximum from L to H isochores? To disclose the role played by each of the potential mechanisms involved in such biased distribution, we carried out a genome-wide analysis of the density of the Alus as a function of their evolutionary age, isochore membership, and intron vs. intergene location. Since Alus depend on the retrotransposase encoded by the LINE1 elements, we also studied the distribution of LINE1 to provide a complete evolutionary scenario. We consecutively check, and discard, the contributions of the Alu/LINE1 competition for retrotransposase, compositional matching pressure, and Alu overrepresentation in introns. In analyzing the role played by unequal recombination, we scan the genome for Alu trimers, a direct product of Alu–Alu recombination. Through computer simulations, we show that such trimers are much more frequent than expected, the observed/expected ratio being higher in L than in H isochores. This result, together with the known higher selective disadvantage of recombination products in H isochores, points to Alu–Alu recombination as the main agent provoking the density shift of Alus toward the GC-rich parts of the genome. Two independent pieces of evidence—the lower evolutionary divergence shown by recently inserted Alu subfamilies and the higher frequency of old stand-alone Alus in L isochores—support such a conclusion. Other evolutionary factors, such as population bottlenecks during primate speciation, may have accelerated the fast accumulation of Alus in GC-rich isochores.     

Alu element-mediated gene silencing

The Alu elements are conserved approximately 300-nucleotide-long repeat sequences that belong to the SINE family of retrotransposons found abundantly in primate genomes. Pairs of inverted Alu repeats in RNA can form duplex structures that lead to hyperediting by the ADAR enzymes, and at least 333 human genes contain such repeats in their 3'-UTRs. Here, we show that a pair of inverted Alus placed within the 3'-UTR of egfp reporter mRNA strongly represses EGFP expression, whereas a single Alu has little or no effect. Importantly, the observed silencing correlates with A-to-I RNA editing, nuclear retention of the mRNA and its association with the protein p54(nrb). Further, we show that inverted Alu elements can act in a similar fashion in their natural chromosomal context to silence the adjoining gene. For example, the Nicolin 1 gene expresses multiple mRNA isoforms differing in the 3'-UTR. One isoform that contains the inverted repeat is retained in the nucleus, whereas another lacking these sequences is exported to the cytoplasm. Taken together, these results support a novel role for Alu elements in human gene regulation.     

Evolutionary Breakpoints in the Gibbon Suggest Association between Cytosine Methylation and Karyotype Evolution

Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15–18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (<150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution.     

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.     

Patterns of segmental duplication in the human genome

We analyzed the completed human genome for recent segmental duplications (size > or = 1 kb and sequence similarity > or = 90%). We found that approximately 4% of the genome is covered by duplications and that the extent of segmental duplication varies from 1% to 14% among the 24 chromosomes. Intrachromosomal duplication is more frequent than interchromosomal duplication in 15 chromosomes. The duplication frequencies in pericentromeric and subtelomeric regions are greater than the genome average by approximately threefold and fourfold. We examined factors that may affect the frequency of duplication in a region. Within individual chromosomes, the duplication frequency shows little correlation with local gene density, repeat density, recombination rate, and GC content, except chromosomes 7 and Y. For the entire genome, the duplication frequency is correlated with each of the above factors. Based on known genes and Ensembl genes, the proportion of duplications containing complete genes is 3.4% and 10.7%, respectively. The proportion of duplications containing genes is higher in intrachromosomal than in interchromosomal duplications, and duplications containing genes have a higher sequence similarity and tend to be longer than duplications containing no genes. Our simulation suggests that many duplications containing genes have been selectively maintained in the genome.     

A novel approach for identifying candidate imprinted genes through sequence analysis of imprinted and control genes

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.     

Structural organization of the human genome: distribution of nucleotides, Alu-repeats and exons in chromosomes 21 and 22]

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (Y waves phi) with a period of about 3 Mbp. Distribution of G + C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G + C than chromosome 22. Both exons and Alu repeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alu repeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alu repeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Alu distribution patterns along the chromosome, the concurrent distribution of Alu repeats in both orientations along the chromosome, and the equal copy numbers for Alu in direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alu repeats belong to parasitic (junk) DNA.     

Inverted Alu dsRNA structures do not affect localization but can alter translation efficiency of human mRNAs independent of RNA editing

With over one million copies, Alu elements are the most abundant repetitive elements in the human genome. When transcribed, interaction between two Alus that are in opposite orientation gives rise to double-stranded RNA (dsRNA). Although the presence of dsRNA in the cell was previously thought to only occur during viral infection, it is now known that cells express many endogenous small dsRNAs, such as short interfering RNA (siRNAs) and microRNA (miRNAs), which regulate gene expression. It is possible that long dsRNA structures formed from Alu elements influence gene expression. Here, we report that human mRNAs containing inverted Alu elements are present in the mammalian cytoplasm. The presence of these long intramolecular dsRNA structures within 3'-UTRs decreases translational efficiency, and although the structures undergo extensive editing in vivo, the effects on translation are independent of the presence of inosine. As inverted Alus are predicted to reside in >5% of human protein-coding genes, these intramolecular dsRNA structures are important regulators of gene expression.     

Genes on human chromosome 19 show extreme divergence from the mouse orthologs and a high GC content

Mutational rates are known to be variable along the mammalian genome but the extent of this non-random fluctuation and their causes are less well understood. Using 5509 human and mouse orthologous genes with known chromosome positions, it is shown here that there are extreme differences in synonymous evolutionary rates between different human chromosomes when distances are measured using maximum-likelihood techniques. In particular, the average synonymous rate of genes located in human chromosome 19 is extremely high (K_s = 1.243 substitutions/site) compared with the average of all genes (K_s = 0.729), and significantly different from all other human chromosomes. When genes are sorted according to mouse chromosomes no such large differences are found. Strikingly, almost all genes of human chromosome 19 have very high GC content in humans but not in the mouse orthologs. More generally, correlation analysis shows that genes with very high GC content in humans have experienced the highest synonymous divergencies from the mouse. It is likely that, in such genes, the known relaxation of the isochore structure in rodents has caused an increased accumulation of synonymous substitutions in the mouse lineage, whereas the regions with the highest GC content in the human genome are accordingly maintained by a strong selective pressure.     

Comparative genomic sequencing reveals a strikingly similar architecture of a conserved syntenic region on human chromosome 11p15.3 (including gene ST5) and mouse chromosome 7

Comparative genomics is a superior way to identify phylogenetically conserved features like genes or regions involved in gene regulation. The comparison of extended orthologous chromosomal regions should also reveal other characteristic traits essential for chromosome or gene function. In the present study we have sequenced and compared a region of conserved synteny from human chromosome 11p15.3 and mouse chromosome 7. In human, this region is known to contain several genes involved in the development of various disorders like Beckwith-Wiedemann overgrowth syndrome and other tumor diseases. Furthermore, in the neighboring chromosome region 11p15.5 extensive imprinting of genes has been reported which might extend to region 11p15.3. The analysis of approximately 730 kb in human and 620 kb in mouse led to the identification of eleven genes. All putative genes found in the mouse DNA were also present in the same order and orientation in the human chromosome. However, in the human DNA one putative gene of unknown function could be identified which is not present in the orthologous position of the mouse chromosome. The sequence similarity between human and mouse is higher in transcribed and exon regions than in non-transcribed segments. Dot plot analysis, however, reveals a surprisingly well-conserved sequence similarity over the entire analyzed region. In particular, the positions of CpG islands, short regions of very high GC content in the 5' region of putative genes, are similar in human and mouse. With respect to base composition, two distinct segments of significantly different GC content exist as well in human as in the mouse. With a GC content of 45% the one segment would correspond to "isochore H1" and the other segment (39% GC in human, 40% GC in mouse) to "isochore L1/L2". The gene density (one gene per 66 kb) is slightly higher than the average calculated for the complete human genome (one gene per 90 kb). The comparison of the number and distribution of repetitive elements shows that the proportion of human DNA made up by interspersed repeats (43.8%) is significantly higher than in the corresponding mouse DNA (30.1%). This partly explains why the human DNA is longer between the landmark genes used to define the orthologous positions in human and mouse.     

Molecular evaluation of an Alu repeat including a polymorphic variable poly(dA) (AluVpA) in the vitamin D binding protein (DBP) gene

We investigated an Alu element at the end of intron 8 of the human vitamin D-binding protein (hDBP, group-specific component, GC) gene that shows a polymorphic poly(A) tail due to a variable number of tandem repeats (AluVpA) forming the 3 end of this member of the most abundant class of short interspersed repeated DNA element (SINES). The Alu element sequence in intron 8 of the GC gene was identical in all three common GC alleles (GC*1F, GC*1S, and GC*2) and could be classified as an Alu-Sa or Alu class-II sequence. The polymerase chain reaction was used to amplify selectively a fragment of about 200 bp containing the identified (TAAA)n repeat from genomic DNA of 188 unrelated human subjects. The size of the amplified products was determined by polyacrylamide gel electrophoresis. Four alleles (named GC-18*6, GC-I8*8, GC-I8*10, and GC-18*11) were found that differed in size by multiples of four nucleotides. The allele frequencies ranged from 0.0053 to 0.8511 and the observed heterozygosity was 26%. The stable inheritance of this polymorphic patterned poly(A) sequence was confirmed by a segregation study of a highly informative family with 19 members. Statistically significant linkage disequilibrium between the AluVpA and the GC isoelectric focusing (IEF) phenotypes was found in a sample of 188 unrelated individuals and delta values were calculated from the observed haplotype distribution.     

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.