A Comparative Analysis of Regulatory Regions of the Transthyretin Gene in the Mouse, Human, and Chimpanzee Genomes

A full genome analysis of differences between the gene expression in the human and chimpanzee brains revealed that the gene for transthyretin, the carrier of thyroid hormones, is differently transcribed in the cerebella of these species. A 7-kbp DNA fragment of chimpanzee was sequenced to identify possible regulatory sequences responsible for the differences in expression. One hundred and thirteen substitutions were found in the chimpanzee sequence in comparison with the human sequence. About 40% of the substitutions were revealed within the repeating elements of the genome; their location and sizes did not differ from those in the corresponding fragments of the human genome, and the nucleotide sequences had a high degree of identity. A comparison of nucleotide sequences of the transthyretin region of human, chimpanzee, and mouse genes revealed substantial differences in the distribution of G + C content along the examined fragment in the human (chimpanzee) and mouse genes and allowed us to localize three sequence tracts with a higher degree of identity in the three species. One of these tracts was located in the promoter region of the gene, and the other two probably determine the specificity of transthyretin gene expression in the liver and brain. One of the conserved tracts of the chimpanzee genome was found to have a single and a triple nucleotide substitution. The triple substitution distinguishes chimpanzees from humans and mice, which have identical sequences of this site. It is likely that these substitutions are responsible for the differences in the expression levels of the transthyretin gene in the human and chimpanzee brains.     

A comparative analysis of regulatory regions of the transthyretin gene in the mouse, human, and chimpanzee genomes

A full genome analysis of differences between the gene expression in the human and chimpanzee brains revealed that the gene for transthyretin, the carrier of thyroid hormones, is differently transcribed in the cerebella of these species. A 7-kbp DNA fragment of chimpanzee was sequenced to identify possible regulatory sequences responsible for the differences in expression. One hundred and thirteen substitutions were found in the chimpanzee sequence in comparison with the human sequence. About 40% of the substitutions were revealed within the repeating elements of the genome; their location and sizes did not differ from those in the corresponding fragments of the human genome, and the nucleotide sequences had a high degree of identity. A comparison of nucleotide sequences of the transthyretin region of human, chimpanzee, and mouse genes revealed substantial differences in the distribution of G + C content along the examined fragment in the human (chimpanzee) and mouse genes and allowed us to localize three sequence tracts with a higher degree of identity in the three species. One of these tracts is located in the promoter region of the gene, and the other two probably determine the specificity of transthyretin gene expression in the liver and brain. One of the conserved tracts of the chimpanzee genome was found to have a single and a triple nucleotide substitution. The triple substitution distinguishes chimpanzees from humans and mice, which have identical sequences of this site. It is likely that these substitutions are responsible for the differences in the expression levels of the transthyretin gene in the human and chimpanzee brains.     

Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.     

Gene diversity of chimpanzee ABO blood group genes elucidated from exon 7 sequences

Human and non-human primate ABO blood group genes show relatively large numbers of nucleotide differences. In this study, we determined exon 7 sequences for 10 individuals of common chimpanzee and for four individuals of bonobo to estimate nucleotide diversities among them. Sequence data showed the existence of chimpanzee specific 9-base deletion in the beginning of the exon 7 coding region. From a phylogenetic network of exon 7 sequences of ABO blood group genes for human, common chimpanzee, bonobo and gorilla, effects of parallel substitutions and/or some kinds of convergent events are inferred in the chimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes, and these values were 0.4% and 0.2%, respectively. These values are higher than that of most human genes.     

Human genomic deletions mediated by recombination between Alu elements

Recombination between Alu elements results in genomic deletions associated with many human genetic disorders. Here, we compare the reference human and chimpanzee genomes to determine the magnitude of this recombination process in the human lineage since the human-chimpanzee divergence approximately 6 million years ago. Combining computational data mining and wet-bench experimental verification, we identified 492 human-specific deletions (for a total of approximately 400 kb) attributable to this process, a significant component of the insertion/deletion spectrum of the human genome. The majority of the deletions (295 of 492) coincide with known or predicted genes (including 3 that deleted functional exons, as compared with orthologous chimpanzee genes), which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees. Overall, we found that Alu recombination-mediated genomic deletion has had a much higher impact than was inferred from previously identified isolated events and that it continues to contribute to the dynamic nature of the human genome.     

Concerted evolution of the primate immunoglobulin alpha-gene through gene conversion.

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (C alpha) genes (chimpanzee C alpha 2, gorilla C alpha 2, and gibbon C alpha 1 and C alpha 2 genes), which made possible the examination of gene conversions in all hominoid C alpha genes. The following three methods were used to detect gene conversions: 1) phenetic tree construction; 2) detection of a DNA segment with extremely low variability between duplicated C alpha genes; and 3) a site by site search of shared nucleotide changes between duplicated C alpha genes. Results obtained from method 1 indicated a concerted evolution of the duplicated C alpha genes in the human, chimpanzee, gorilla, and gibbon lineages, while results obtained from method 2 suggested gene conversions in the human, gorilla, and gibbon C alpha genes. With method 3 we identified clusters of shared nucleotide changes between duplicated C alpha genes in human, chimpanzee, gorilla, and gibbon lineages, and in their hypothetical ancestors. In the present study converted regions were identified over the entire C alpha gene region excluding a few sites in the coding region which have escaped from gene conversion. This indicates that gene conversion is a general phenomenon in evolution, that can be clearly observed in non-functional regions.     

Human-specific amino acid changes found in 103 protein-coding genes

We humans have many characteristics that are different from those of the great apes. These human-specific characters must have arisen through mutations accumulated in the genome of our direct ancestor after the divergence of the last common ancestor with chimpanzee. Gene trees of human and great apes are necessary for extracting these human-specific genetic changes. We conducted a systematic analysis of 103 protein-coding genes for human, chimpanzee, gorilla, and orangutan. Nucleotide sequences for 18 genes were newly determined for this study, and those for the remaining genes were retrieved from the DDBJ/EMBL/GenBank database. The total number of amino acid changes in the human lineage was 147 for 26,199 codons (0.56%). The total number of amino acid changes in the human genome was, thus, estimated to be about 80,000. We applied the acceleration index test and Fisher's synonymous/nonsynonymous exact test for each gene tree to detect any human-specific enhancement of amino acid changes compared with ape branches. Six and two genes were shown to have significantly higher nonsynonymous changes at the human lineage from the acceleration index and exact tests, respectively. We also compared the distribution of the differences of the nonsynonymous substitutions on the human lineage and those on the great ape lineage. Two genes were more conserved in the ape lineage, whereas one gene was more conserved in the human lineage. These results suggest that a small proportion of protein-coding genes started to evolve differently in the human lineage after it diverged from the ape lineage.     

Complex events in the evolution of the haptoglobin gene cluster in primates

Southern blot analyses of genomic DNA show that new world monkeys have only one haptoglobin gene but that chimpanzees, gorillas, orangutans, and old world monkeys have three. Humans have two: haptoglobin (Hp) and haptoglobin-related (Hpr). These observations suggest that a triplication of the haptoglobin locus occurred after the divergence of the new world monkeys, followed by a deletion of one locus in humans. To investigate these events, we have cloned the haptoglobin gene cluster in chimpanzee. The organization of the Hp and Hpr genes in chimpanzees is the same as in humans, including a retrovirus-like sequence in the first intron of Hpr. The third gene, which we name Hpp for haptoglobin primate, is 16 kilobases downstream of Hpr. A second copy of the retrovirus-like sequence occurs between Hpr and Hpp. The nucleotide sequence of the chimpanzee Hpp gene suggests that it may code for a functional protein, but the chimpanzee Hpr gene has a single base deletion in exon 5 that causes a frameshift. Comparison of the human and chimpanzee sequences suggests that the human Hpr gene was generated by a homologous unequal crossover between ancestral Hpr and Hpp genes. The crossover point lies within a 1.3-kilobase region containing exon 5 and 500 nucleotides 3' to the genes, but the exact point is obscured by a subsequent gene conversion event.     

Intragene higher order repeats in neuroblastoma breakpoint family genes distinguish humans from chimpanzees

Much attention has been devoted to identifying genomic patterns underlying the evolution of the human brain and its emergent advanced cognitive capabilities, which lie at the heart of differences distinguishing humans from chimpanzees, our closest living relatives. Here, we identify two particular intragene repeat structures of noncoding human DNA, spanning as much as a hundred kilobases, that are present in human genome but are absent from the chimpanzee genome and other nonhuman primates. Using our novel computational method Global Repeat Map, we examine tandem repeat structure in human and chimpanzee chromosome 1. In human chromosome 1, we find three higher order repeats (HORs), two of them novel, not reported previously, whereas in chimpanzee chromosome 1, we find only one HOR, a 2mer alphoid HOR instead of human alphoid 11mer HOR. In human chromosome 1, we identify an HOR based on 39-bp primary repeat unit, with secondary, tertiary, and quartic repeat units, fully embedded in human hornerin gene, related to regenerating and psoriatric skin. Such an HOR is not found in chimpanzee chromosome 1. We find a remarkable human 3mer HOR organization based on the ~1.6-kb primary repeat unit, fully embedded within the neuroblastoma breakpoint family genes, which is related to the function of the human brain. Such HORs are not present in chimpanzees. In general, we find that human-chimpanzee differences are much larger for tandem repeats, in particularly for HORs, than for gene sequences. This may be of great significance in light of recent studies that are beginning to reveal the large-scale regulatory architecture of the human genome, in particular the role of noncoding sequences. We hypothesize about the possible importance of human accelerated HOR patterns as components in the gene expression multilayered regulatory network.     

Selective sweeps in the human genome: a starting point for identifying genetic differences between modern humans and chimpanzees

Despite more than a century of interest in the evolution ofhumans from our close relatives the great apes, the genes responsiblefor phenotypic differences between humans and chimpanzees haveremained elusive. Sequencing of the chimpanzee genome is expectedto identify some 42 million nucleotide differences between humansand chimpanzee. How can we identify the small proportion ofthese differences which are the essential elements of beinghuman? We have analyzed the draft human genome to find regionswhich may have experienced recent strong selection in the humanline. Included in the identified regions are several genes forneural development and function, skeletal development, and fatmetabolism. These observations provide a starting point in thesearch to identify the salient genetic differences between modernhumans and our immediate hominid ancestors. Strong directional selection for a favorable new allele cancause     

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.     

Gene diversity of chimpanzee ABO blood group genes elucidated from intron 6 sequences

The human and nonhuman primate ABO blood group gene shows relatively large numbers of nucleotide differences around the exon 7 region. In this study we determined intron 6 sequences for 9 alleles of common chimpanzee and for 3 alleles of bonobo to estimate nucleotide diversities among them. Sequence length polymorphisms are observed in this region as a repeat appears one to five times. From a phylogenetic network of intron 6 sequences of ABO blood group genes for humans, common chimpanzee, and bonobo, parallel substitutions and/or some kinds of convergent events are predicted in the chimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes; these values were 0.219% and 0.208%, respectively.     

The chimpanzee GH locus: composition, organization, and evolution

In most mammals the growth hormone (GH) locus comprises a single gene expressed primarily in the anterior pituitary gland. However, in higher primates multiple duplications of the GH gene gave rise to a complex locus containing several genes. In man this locus comprises five genes, including GH-N (expressed in pituitary) and four genes expressed in the placenta, but in other species the number and organization of these genes vary. The situation in chimpanzee has been unclear, with suggestions of up to seven GH-like genes. We have re-examined the GH locus in chimpanzee and have deduced the complete sequence. The locus includes five genes apparently organized in a fashion similar to that in human, with two of these genes encoding GH-like proteins, and three encoding chorionic somatomammotropins/placental lactogens (CSHs/PLs). There are notable differences between the human and chimpanzee loci with regard to the expressed proteins, gene regulation, and gene conversion events. In particular, one human gene (hCSH-L) has changed substantially since the chimpanzee/human split, potentially becoming a pseudogene, while the corresponding chimpanzee gene (CSH-A1) has been conserved, giving a product almost identical to the adjacent CSH-A2. Chimpanzee appears to produce two CSHs, with potentially differing biological properties, whereas human produces a single CSH. The pattern of gene conversion in human has been quite different from that in chimpanzee. The region around the GH-N gene in chimpanzee is remarkably polymorphic, unlike the corresponding region in human. The results shed new light on the complex evolution of the GH locus in higher primates.     

Random nucleotide substitutions in primate nonfunctional gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis

Humans and other primates have no functional gene for L-gulono-gamma-lactone oxidase that catalyzes the last step of L-ascorbic acid biosynthesis. The 164-nucleotide sequence of exon X of the gene was compared among human, chimpanzee, orangutan, and macaque, and it was found that nucleotide substitutions had occurred at random throughout the sequence with a single nucleotide deletion, indicating that the primate L-gulono-gamma-lactone oxidase genes are a typical example of pseudogene.     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of sequence from the human genome and ~15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.     

Functional analysis of human and chimpanzee promoters

Background  

It has long been argued that changes in gene expression may provide an additional and crucial perspective on the evolutionary differences between humans and chimpanzees. To investigate how often expression differences seen in tissues are caused by sequence differences in the proximal promoters, we tested the expression activity in cultured cells of human and chimpanzee promoters from genes that differ in mRNA expression between human and chimpanzee tissues.