*Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom;
University Department of Paediatrics, Oxford, United Kingdom
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Abstract |
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Introduction |
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There are three known mechanisms by which a transposition event might affect gene regulation: (1) The insertion may disrupt existing regulatory elements (Wallace et al. 1991
); (2) the regulatory sequences of the transposable element itself may act on genes that are close to the site of insertion (Willoughby, Vilalta, and Oshima 2000)
; or (3) the insertion may simply provide an additional sequence within which gene regulatory elements can subsequently evolve (Hambor et al. 1993
; Britten 1996
).
This study considers a further proposed mechanism, whereby a transposable element comobilizes an adjacent regulatory element to a new genomic location (Britten and Davidson 1969, 1971
; Moran, DeBerardinis, and Kazazian 1999
). If such a mechanism existed, it would be of considerable evolutionary significance because unlike sequences which have arisen through the accumulation of mutations over millions of years, a regulatory element introduced in this manner would have immediate functional potential. When coupled with natural selection, this might favor the evolution of complex gene regulatory networks.
Specifically, we examine the evolution of a complex regulatory element in the human interferon- promoter region. Interferon-
is a cytokine secreted by activated lymphocytes, acting as a first line of host defense against infectious agents by activating macrophages as well as playing a critical role in antibody production and cytotoxic T-cell function (Thèze 1999
). In humans, the expression of interferon-
is regulated by a 20-bp segment of DNA containing binding sites for the transcription factors NFAT and NF-
B, both of which have been shown to contribute to transcriptional activation of the gene (Sica et al. 1997
). The present study was prompted by the observation that this segment of DNA lies close to the boundary of an Alu insertion.
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Materials and Methods |
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Detection of Alu Insertion by PCR
Forward and reverse primers (forward primer 5'-ACT CAC AAT CAT ATA GCT AG-3', reverse primer 5'-AAG TCT CCT GAG GAT TAC GT-3') were designed to amplify across both the MER33 and the AluSg repetitive elements. Amplification was performed in a 15-µl reaction with 4.0 mM MgCl2, 200 µM of each dNTP, 1 x Opti buffer (Bioline), 3.1 µM of each primer, 0.75 units of BIO-X-ACT Taq polymerase (Bioline), and 1 ng of genomic DNA. These primers amplify a specific 1.5-kb fragment from human genomic DNA using the following cycling conditions: 94°C 2 min; then 10 cycles of 94°C 15 s, 57°C 30 s, 68°C 2 min, followed by 20 cycles of 94°C 15 s, 57°C 30 s, 68°C 2 min plus 10 s/cycle; and a final 5 min extension at 68°C. Species that do not have the AluSg insertion amplify a 1.2-kb fragment. Genomic DNA samples from one human, two Common Chimpanzees (Pan troglodytes), one Gorilla (Gorilla gorilla), two Orangutans (Pongo pygmaea), one Lar Gibbon (Hylobates lar), two Sulawesi Macaques (Macaca nigra), one Patas (Erythrocebus patas), two Black-handed Spider Monkeys (Ateles paniscus), and one Golden-headed Lion Tamarin (Leontopithecus chrysomelas) were amplified and visualized under ultraviolet light after electrophoresis through a 1% agarose gel with ethidium bromide staining.
Sequencing the Interferon- Promoter in Nonhuman Primates
Samples
All 13 of the individuals from the PCR experiment plus one Hanuman Langur (Presbytis entellus) and one Abyssinian Colobus (Colobus guereza) were sequenced.
Sequencing Catarrhines
A second round PCR using forward primer 5'-ACT CAC AAT CAT ATA GCT AG-3' and reverse primer 5'-AAT GAC CAG AAA GCA AGG AAA G-3' amplified a 1,053-bp fragment under the following reaction conditions: a 15-µl reaction with 2.5 mM Mg, 20 µM of each dNTP, 15 mM Tris-HCl, 50 mM KCl, pH 8.0, 1.0 µM of each primer, 0.5 units of Taq Gold polymerase (Perkin-Elmer), and 1 µl of a 1:20 dilution of the 1.5-kb PCR product described earlier. Thermocycling conditions were: 94° 10 min followed by 30 cycles of 94° 30 s, 54° 30 s, 72° 30 s, and a final extension for 5 min at 72°. Dye-terminator sequencing was performed as prescribed by Perkin-Elmer using a nested forward primer: 5'-TGA GAC GGA ATC TAC TCT GT-3'.
Sequencing Platyrrhines
The second round PCR was the same as for the catarrhines but produced a 750-bp fragment. A nested primer (5'-GTC CTT CAT CAG AGT TGG TTA G-3') was used in a dye-terminator sequencing reaction under conditions prescribed by Perkin-Elmer, except for the annealing temperature which was 58°C.
Confirmatory Resequencing
All samples were reamplified using M13-tailed primers; PCR products were purified using QiaQuick spin columns (Qiagen) and sequenced using the Perkin-Elmer dye-primer chemistry. No discrepancies from the original sequence were found.
Sequence Analysis
Sixteen different sequences (GenBank AF323472AF323487, alignment available from PopSet 13447766) representing nine catarrhine species and two platyrrhine species were aligned using ClustalX (Thompson et al. 1997
). To allow for the Alu insertion in the catarrhines, the gap extension penalty was reduced when aligning the platyrrhine sequences. An alignment of the nine catarrhine species provided 300 bp of sequence that spans the NF-
BNFAT regulatory element. Variable sites were counted through a 10-bp sliding window and presented graphically to illustrate the clustering of mutations between positions -778 and -795.
Electrophoretic Mobility Shift Assay
Oligonucleotide probes were radiolabeled with [a-32P]-dCTP (Amersham Pharmacia Biotech, see table 1
for sequences of probes). COS-7 cells were transfected with CMV-p50expressing and CMV-p65expressing constructs, and total protein extracts were prepared by lysing cells in lysis buffer (20 mM Tris-Cl, pH 8.0, 300 mM NaCl, 0.1% NP-40, 10% glycerol) supplemented with protease inhibitors (Boehringer Mannheim). The binding reaction contained 12 mM HEPES, pH 7.8, 80100 mM KCl, 1 mM EDTA, 1 mM EGTA, 12% glycerol, and 0.5 mg of poly dI-dC (Amersham Pharmacia Biotech). Protein extracts (14 µg) were mixed in an 8-µl reaction with 0.20.5 ng of radiolabeled probe (15 x 104 CPM) and incubated at room temperature for 10 min. The reaction was analyzed by electrophoresis in a nondenaturing 5% polyacrylamide gel at 4°C in 0.5 x TBE buffer. Where indicated, gels were quantified using the Phosphorimager (Molecular Dynamics).
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Results |
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To obtain a more precise estimate of when the Alu insertion occurred, we performed PCR amplification in various primate species, using primers based on human sequence flanking the MER element (fig. 1 ). Apes (gibbon, orangutan, gorilla, chimpanzee, human) and Old World monkeys (patas, macaque) gave a 1.5-kb PCR product, corresponding to the predicted size if both MER and AluSg elements were present. In contrast, New World monkeys (spider monkey and lion tamarin) gave a 1.2-kb PCR product corresponding to the predicted size if the AluSg element was missing. Sequencing of the PCR products confirmed that this AluSg insertion occurred in the catarrhine lineage after the divergence of catarrhines and platyrrhines, approximately 2234 MYA (fig. 2 ) (GenBank: AF323472AF323487).
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To determine how this compares with interspecific nucleotide diversity in the surrounding region, we counted variable sites between -886 and -593 nt through a 10-bp sliding window. A distinct peak of variation was observed around -790 nt, i.e., the point where the AluSg sequence abuts the NF-B binding site (fig. 4
). Across the 295-bp sequence analyzed, there were 50 variable sites of which 11 lay within a 16-bp segment (-793 to -778) which includes the 10-bp NF-
B binding site (-788 to -779). To determine whether this degree of clustering was likely to happen by chance, a computer simulation was designed to uniquely place 50 mutations randomly across 295 bases. The observed clustering of variable sites occurred nine times in 200,000 simulations. Thus, it appears that the NFAT binding site was inserted with the Alu element and has subsequently been preserved, whereas the adjacent NF-
B binding site has arisen through base substitutions subsequent to insertion.
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Comparison of the human and macaque sequences with the NF-B consensus motif suggests that a critical evolutionary event may have been a double transversion at the nucleotides corresponding to positions 3 and 4 of the NF-
B site. The clade containing macaque, patas, colobus, and langur has CC at this position, whereas the humans, gorilla, chimpanzees, orangutans, and gibbon all have AA. To examine the specific effect of this double transversion on binding affinity, we synthesized an oligoduplex that was identical to the human sequence apart from these two nucleotides which were changed from AA to CC. This resulted in almost complete loss of p65-p50 and p50-p50 binding (fig. 5
).
Analysis of the human interferon- promoter region has revealed that three single nucleotide polymorphisms lie extremely close to the NFAT and NF-
B binding sites (J. Hull et al, unpublished data). They are a C to G substitution at -765 nt (flanking the 3' end of the NFAT binding site), a C to T substitution at -778 nt (flanking the 3' end of the NF-
B binding site), and a C to T substitution at -793 nt (5 positions 5' of the NF-
B binding site) (fig. 3B
). Table 2
summarizes the variation detected at these three positions by dideoxy sequencing of genomic DNA from 108 individuals drawn from two human populations (36 Europeans and 72 West Africans). The -793T allele was found in seven West Africans and no Europeans; the -778T allele was found in one West African and no Europeans; and the -765G allele was found in five Europeans and no West Africans.
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Discussion |
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An important conclusion is that Alu, the most ubiquitous family of transposable elements, can modify the regulation of a gene by carrying with it an intact transcription factor binding site from another part of the genome. Although such a mechanism has been proposed it has not previously been observed in nature. This finding lends weight to the hypothesis that transposable elements have served to disperse transcription factor binding sites across the genome, thereby facilitating the evolution of regulatory networks.
After the NFAT binding site was inserted into the interferon- promoter region, the adjacent sequence evolved into a binding site for NF-
B. Different primate species show a remarkable degree of nucleotide diversity in and around the NF-
B binding site. On the basis of sequence comparisons and analysis of DNA-protein interactions, it appears that the critical event leading to NF-
B binding was a CC to AA double transversion that occurred in the ancestor to the Apes (fig. 2
). Our evolutionary model (fig. 6
) suggests that the first NF-
B binding sequence has been preserved in gorilla and human, whereas subsequent mutations in orangutan and chimpanzee have led to decreased binding affinity. This raises the possibility that the capacity to bind NF-
B at this site may have been subjected to evolutionary pressures.
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It has been suggested that repetitive DNA may facilitate the dispersion of regulatory elements, allowing natural selection to fashion new networks from existing regulatory sequences (Britten and Davidson 1971
). Previous examples have shown that transposable elements may (1) disrupt existing regulatory mechanisms, (2) introduce new regulatory elements intrinsic to their consensus sequence, and (3) provide relatively unconstrained sequence for the evolution of transcription factor binding sites through base substitution. Our findings indicate that Alu, the most ubiquitous family of transposable elements, is capable of comobilizing regulatory elements extrinsic to the Alu sequence itself. It remains to be seen whether this mechanism is widespread. If so, it may have acted together with natural selection as a force for the evolution of complex regulatory networks.
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Acknowledgements |
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Footnotes |
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Address for correspondence and reprints: Hans Ackerman, 64 Linnaean Street, #572, Harvard University, Cambridge, Massachusetts 02138. ackerman{at}fas.harvard.edu
.
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References |
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