Differential distribution of a SINE element in the Entamoeba histolytica and Entamoeba dispar genomes: Role of the LINE-encoded endonuclease
- Vandana Kumari†1,
- Rahul Sharma†1,
- Vijay P Yadav1,
- Abhishek K Gupta1,
- Alok Bhattacharya2 and
- Sudha Bhattacharya1Email author
© Kumari et al; licensee BioMed Central Ltd. 2011
Received: 27 January 2011
Accepted: 25 May 2011
Published: 25 May 2011
Entamoeba histolytica and Entamoeba dispar are closely related protistan parasites but while E. histolytica can be invasive, E. dispar is completely non pathogenic. Transposable elements constitute a significant portion of the genome in these species; there being three families of LINEs and SINEs. These elements can profoundly influence the expression of neighboring genes. Thus their genomic location can have important phenotypic consequences. A genome-wide comparison of the location of these elements in the E. histolytica and E. dispar genomes has not been carried out. It is also not known whether the retrotransposition machinery works similarly in both species. The present study was undertaken to address these issues.
Here we extracted all genomic occurrences of full-length copies of EhSINE1 in the E. histolytica genome and matched them with the homologous regions in E. dispar, and vice versa, wherever it was possible to establish synteny. We found that only about 20% of syntenic sites were occupied by SINE1 in both species. We checked whether the different genomic location in the two species was due to differences in the activity of the LINE-encoded endonuclease which is required for nicking the target site. We found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. Hence the differential distribution of SINEs in these species is not likely to be influenced by the endonuclease. Further we found that the physical properties of the DNA sequences adjoining the insertion sites were similar in both species.
Our data shows that the basic retrotransposition machinery is conserved in these sibling species. SINEs may indeed have occupied all of the insertion sites in the genome of the common ancestor of E. histolytica and E. dispar but these may have been subsequently lost from some locations. Alternatively, SINE expansion took place after the divergence of the two species. The absence of SINE1 in 80% of syntenic loci could affect the phenotype of the two species, including their pathogenic properties, which needs to be explored.
Transposable elements are found in the genomes of almost all organisms, and are of ancient origin. Their ability to insert into new genomic locations makes them potent agents of phenotypic change, including various known pathologies [1–3]. Transposons are also found in parasites, for example the human enteric pathogen, Entamoeba histolytica, a unicellular eukaryote contains three families of the autonomous non long terminal repeat (LTR) retrotransposon called EhLINE and its nonautonomous partner EhSINE [4–8]. The genome of the morphologically indistinguishable sibling species Entamoeba dispar, (which resides in the human colon but is nonpathogenic), also contains three families of EdLINEs and their partner EdSINEs [6, 7, 9]. It would be of interest to know whether these retrotransposons have any influence on pathogenesis-related gene expression.
LINEs and SINEs are known to influence the expression of neighboring genes by a variety of mechanisms, for example by providing alternative promoters, splicing and polyadenylation sites and by heterochromatinization [10–13]. For this reason it is important to investigate whether these elements are located at syntenic positions in the E. histolytica and E. dispar genomes. Earlier investigations with a limited number of genomic loci showed that the sites occupied by EhSINE1 in the E. histolytica genome were empty at homologous regions in E. dispar and conversely the sites occupied by EdSINE1 in the E. dispar genome were empty in E. histolytica. Although an exhaustive genome-wide survey of LINEs and SINEs in Entamoeba species has been reported , a genome-wide comparison of the occupancy of these elements at syntenic loci in E. histolytica and E. dispar has not been carried out. Here we present results of a genome-wide comparison of EhSINE1 and EdSINE1 in the two genomes. In addition we address the question whether the differences in genomic locations of retrotransposons in these two organisms could be due to inherent differences in the retrotransposition machinery, particularly in the properties of the LINE-encoded endonuclease. Target primed reverse transcription is the mechanism by which non-LTR retrotransposons insert in the genome . Since retrotransposition is initiated by the element-encoded endonuclease (EN) making a nick at the bottom strand of the site of insertion, an important determinant of target site specificity could be the preferred nucleotide sequences recognized by the EN. We have earlier shown that the EhLINE1-encoded EN (Eh EN) nicks preferentially at the consensus sequence 5'-GTATT-3', between A-T and T-T. Here we have investigated whether the endonuclease domain encoded by EdLINE1 (Ed EN) has a different target site specificity which could account for the lack of synteny in the location of SINEs in the two genomes.
Comparative Analysis of E. histolytica and E. dispar with respect to the occupancy of SINE1 elements
Since the Entamoeba genome has not yet been assembled completely for any species, we have done all of our comparative analysis on the basis of SINE1 elements located on the scaffolds. The genome sequence of E. histolytica having 1529 scaffolds and E. dispar having 12258 scaffolds was downloaded [NCBI:AAFB00000000, NCBI:AANV00000000]. A total of 393 EhSINE1 and 302 EdSINE1 elements having lengths greater than 450 bp were located. The EhSINE1 sequences were from Huntley et al  and the EdSINE1 sequences were from the feature table file of E. dispar (updated on Dec. 8, 2008). For genes flanking the EhSINE1 sequences the feature table file of E. histolytica (updated on April 17, 2008) was used. These were downloaded from NCBI and the genes upstream and downstream of SINE1 elements in each of the species were located using perl coding. Finally the orthologues of these genes were searched in the other species using BLAST . The syntenic loci were checked to see whether SINE1 was present there in both species. To check the homology at Scaffold level we used GATA, a graphic alignment tool for comparative sequence analysis . Syntenic loci were further examined to check for the presence of SINE in both species.
DNA sequence features of the SINE1 insertion sites were computed by extracting flanking 80 bp sequences (40 bp upstream and downstream) from the respective genomic sites in E. histolytica and E. dispar, using perl scripting. "DNA SCANNER" was used as previously described  to calculate the DNA parameters, which include T rule, bendability, propeller twist, stacking energy, duplex stability, DNA denaturation, protein-induced deformability, nucleosomal positioning and bending energy .
Construction and Cloning of Ed EN
The consensus amino acid sequences of the E. dispar and E. histolytica endonuclease domains, Ed EN and Eh EN respectively were compared. A total of 23 amino acid positions were different of which 12 amino acid residues were changed in the Eh EN sequence  and 11 amino acid residues (synonymous) were left unchanged to obtain the Ed EN sequence. The desired mutations were introduced either by means of overlapping PCR (Additional file 1 Figure S1) or by site-directed mutagenesis. The primer pairs used are listed (Additional file 2 Table S1). The Ed EN sequence thus obtained (782 bp) was cloned into the Eco RI-Not I site of pET30(b) vector (Novagen) to yield the pET-Ed-EN construct.
Expression and purification of recombinant Ed EN
This was done essentially as described for Eh EN . The pET-Ed-EN construct was transformed in Escherichia coli BL-21 (DE3). Cells were grown in 200 ml of Luria Broth at 30°C to OD600 of 0.6. For induction of hexa-His-tagged Ed EN, IPTG was used to a final concentration of 0.5 mM and the cells were further incubated for 2 hour. The protein was purified by Ni2+-nitrilotriacetic acid-agarose (Qiagen) affinity chromatography and eluted with 250 mM of imidazole. The eluted fractions were checked for the protein by resolving on a 12% SDS PAGE; these fractions were pooled, dialyzed and stored at -20°C. The enzyme activity of Ed EN was measured under the same conditions used for Eh EN, i.e. pH 7.0, at 37°C, and at Mg2+ and NaCl concentrations of 10 and 100 mM respectively .
The whole cell lysate of induced and uninduced BL-21 (DE3) cells was resolved on 12% SDS PAGE and transferred to PVDF membrane by semidry transfer method according to manufacturer's instructions (Bio-Rad). The membrane was blocked overnight with 5% skimmed milk in PBS-T (Phosphate buffer saline with 0.1% Tween 20) and subsequently incubated with anti-His antibody or anti-Eh EN antibody for 1 hour followed by three times washing with PBS-T. The membrane was further incubated with horseradish peroxidase-conjugated secondary antibody and again washed with PBS-T thrice. The protein was detected by the Chemiluminescent HRP Substrate (Millipore).
Nicking assay of radiolabeled 176 bp substrate
The 176 bp DNA fragment was radiolabeled as described earlier . The labeled DNA was resolved on 6% native gel, the band was visualized by ethidium bromide staining, excised from the gel and eluted by crush and soak method . The nicking assay was carried out as described for Eh EN and products were resolved by denaturing electrophoresis on 6-8% polyacrylamide gels containing 7 M Urea . The gels were dried and autoradiographed in the PhosphorImager (Fujifilm).
Nicking assay of pBS DNA
Supercoiled pBluescript (pBS) plasmid DNA was purified by plasmid purification kit (Qiagen). 2 nM of purified Ed EN was incubated with 2-75 nM of supercoiled pBS DNA. The reaction was performed under the conditions employed for Eh EN , at 37°C for 8 minutes and was stopped with 25 mM EDTA. Products were separated on 0.8% agarose gel containing 0.5 μg of ethidium bromide/ml. Under this condition the supercoiled DNA migrated fastest, followed by linear and open circular form. The intensity of supercoiled DNA band was measured as a function of time, which gave the measure of the disappearance of supercoiled DNA. Quantification was done by densitometry; the kinetic constants Vmax, Km and kcat were determined as described . The data obtained were the average of three independent determinations.
Results and Discussion
Comparative analysis of EhSINE1-containing regions of E. histolytica genome and syntenic regions of E. dispar
For 213 EhSINE1-containing loci of E. histolytica, syntenic loci could not be found in E. dispar for the following reasons. In many of these cases the scaffolds containing these loci were composed entirely of repeats (83 loci), tRNA genes and repeats (7 loci) or pseudogenes and repeats (9 loci). In 114 cases synteny could not be determined either because there were multiple copies of homologous genes, or homologous genes were located on multiple scaffolds, or there was a single gene in the scaffold.
Comparative analysis of EdSINE1-containing regions of E. dispar genome and syntenic regions of E. histolytica
A total of 302 full-length SINE1 elements (length greater than 450 bp) were identified in E. dispar by genome sequence analysis. Syntenic regions corresponding to each of the EdSINE1-containing loci were located in E. histolytica and the presence or absence of any of the EhSINEs (EhSINE1, 2, 3) was determined as described above. Of the 302 loci, syntenic regions could be predicted with certainty for 127 loci (Figure 1B). Of these, SINEs were absent in E. histolytica at 73 loci, were present at 19 loci and their presence or absence could not be determined at 35 loci. Amongst the 73 loci where SINEs were absent, no repeat element of any type was found at 62 loci (Additional file 5 Figure S20), while LINE sequences were found at 11 loci. Amongst the 19 loci that contained a SINE, 18 had EhSINE1 (as scored above) while 1 had EhSINE2. Amongst the 35 loci where presence or absence of SINE could not be established, in 23 cases the scaffold ended within the locus in E. histolytica, and in 12 cases the homology was restricted to genes on one side of the SINE while there was no homology on the other side.
For 175 EdSINE1-containing loci, syntenic loci could not be found in E. histolytica for the reasons mentioned in the previous section. In 75 cases the E. dispar loci were composed entirely of repeats while in 100 cases synteny could not be determined either because there were multiple copies of homologous genes, or homologous genes were located on multiple scaffolds, or there was a single gene in the scaffold.
Sequence alignment of syntenic loci
From the above data it is clear that only in about 20% of cases where presence or absence of SINE1 could be established at syntenic loci, are SINE elements located in the same intergenic region (although at different insertion points) in both species. In >80% of these loci SINE1 was not found at the same location in both species. Since the elements in the two species have a common lineage and are closely related, what possible factors might account for these differences? According to the Target primed Reverse Transcription model, retrotransposition is initiated by the LINE-encoded Endonuclease (EN) nicking the bottom strand of the target site . Hence it is reasonable to believe that the sequences preferentially nicked by the EN could be the preferred insertion sites of the retrotransposon, and the behavior of EN might influence the choice of target site of a non LTR retrotransposon. Since the Eh EN and Ed EN differ from each other at many amino acid positions (as shown below), it is possible that the two enzymes may have evolved different recognition specificities. To establish this we studied the properties of the EdLINE1-encoded EN and compared it with EhLINE1-encoded EN.
Cloning and expression of the EdLINE1 endonuclease (Ed EN) polypeptide
Kinetics of the Ed EN-catalyzed reaction with pBS supercoiled DNA substrate under steady-state conditions
Nicking site sequence preference of Ed EN
DNA structural features of E. dispar and E. histolytica SINE1 insertion sites
We checked whether the intergenic regions at loci where SINEs were found in both genomes shared greater sequence similarity compared with loci where SINEs did not occur in both genomes. However this was not found to be the case. Intergenic regions in both sets of loci showed overall sequence similarity in the range of 75-90%. E. histolytica and E. dispar are very closely related sibling species  which were in fact classified as a single species until they were re-described as two separate species . Their close relationship is also evident from phylogeny based on LINE-derived RT sequences. This analysis showed that all three families of LINEs and SINEs already existed in the common ancestor before E. histolytica and E. dispar separated into two distinct species . The great similarity between the two ENs of E. histolytica and E. dispar, as found in this study, again shows that the basic retrotransposition machinery is highly conserved in these sibling species. It is therefore possible that SINEs may indeed have occupied all of the potential insertion sites in the genome of the common ancestor of E. histolytica and E. dispar but many of the inserted elements may have been preferentially lost in each genome as the two species diverged from each other. Indeed the differential loss of retrotransposons from specific loci might have contributed to speciation [25, 26]. On the other hand it may be possible that SINE expansion took place after the divergence of the two species, and only a sub set of the potential insertion sites in the E. histolytica and E. dispar genomes are currently occupied. In that case one may expect that each of these extant genomes may possess a large number of 'empty' sites where SINEs could potentially insert in future. A hallmark of retrotransposition is the appearance of target site duplication (TSD) following the insertion of a new element. In syntenic loci an unoccupied site is expected to have one copy of the TSD which is duplicated in the occupied site. We checked for TSD sequences in E. dispar unoccupied sites corresponding to the syntenic E. histolytica occupied sites. We randomly picked 75 loci of E. histolytica, where SINE1 is absent in E. dispar and looked for matches with the TSD at each locus. At 19 loci we found very good match with the TSD sequence (matched length greater than 15 bp, and sequence identity greater than 85%). The occurrence of close matches of TSD sequences in the syntenic loci of E. dispar suggests that potential empty sites may exist where future retrotransposition events could take place.
Our data show that the LINE-encoded endonucleases, Eh EN and Ed EN are essentially very similar, both in their kinetic properties and in their substrate sequence specificity. The DNA structural features of SINE-occupied sites in E. histolytica and E. dispar are also similar. However the elements do not insert at the same sites in the two species. Even in the 20% cases where the elements are located in the same intergenic regions in the two genomes the exact point of insertion is not the same. It is possible that, despite the Eh EN and Ed EN being very similar, the complete retrotransposition machinery consisting of the ribonucleoprotein assembly of LINE-encoded ORF1, ORF2 and the SINE transcript might function differently in the two species, thus leading to the observed differences. Since the elements are very closely related, these differences are likely to be the result of subtle changes that got established after the divergence of the two species. An experimental test of these functional changes requires the complete assembly of ORF1 and ORF2 of EdLINE1. On the other hand the possibility exists that the retrotransposition machineries in the two species are in fact identical and the observed differences are due to the stochastic nature of the insertion process. The number of potential insertion sites of these elements is likely to be large since they do not insert at specific sequences. The only known specificity of the process is that the elements in both E. histolytica and E. dispar insert only in intergenic regions and not within genes. Within intergenic regions they insert near T-rich stretches [18, 19]. As discussed above, in this scenario one would expect to find a large number of 'empty' sites in each genome which could be targets of future insertions. This needs to be experimentally tested. A further possibility is that common insertions did indeed occur in the two genomes, but these were subsequently lost in a differential manner due to selection pressure. If the loss of SINE copies from specific locations conferred a growth advantage to either species, these elements could well have shaped the physiological evolution of the two species, including their virulence properties.
Whatever may have been the mechanisms and processes that determined the positioning of SINEs, the resultant effect is that SINEs occupy different intergenic locations in the two genomes. It is well recognized that LINEs and SINEs can modulate the expression of genes in their vicinity by providing alternative promoters, splicing and polyadenylation sites and by heterochromatinization [10–13]. The absence of SINE1 in >80% of syntenic loci in the extant genomes of E. histolytica and E. dispar could result in differential expression of genes at these loci. This could profoundly influence the phenotype of the two species, which needs to be explored. Recently Lorenzi et al.  in their reannotation and analysis of the E. histolytica genome have listed a large number of protein families showing high association with repetitive elements. Though the top three families which are associated with TEs 100% of the time are hypothetical proteins, important known protein families are also listed. These include gal/gal Nac lectin, hsp70 BspA-like surface protein family and AIG family associated with resistance to bacteria. Further analysis of a similar nature with E. dispar genome will give interesting information on the possible contribution of TEs in regulating the expression of important genes that may influence pathogenesis.
This work was supported by a grant to SB from Indian Council of Medical Research and by fellowship from Council of Scientific and Industrial Research, India to VK and AKG.
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