Evolutionary Diversification of DYX1C1 Transcripts Via an HERV-H LTR Integration Event

Genes Genet. Syst. (2011) 86, p. 277–284 Evolutionary diversification of DYX1C1 transcripts via an HERV-H LTR integration event

Yun-Ji Kim1, Jae-Won Huh2, Dae-Soo Kim3, Kyudong Han4, Hwan-Mook Kim5 and Heui-Soo Kim1* 1Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea 2National Primate Research Center (NPRC), KRIBB, Ochang, Chungbuk 363-883, Republic of Korea 3Korean BioInformation Center, KRIBB, Daejeon 305-806, Republic of Korea 4Department of Nanobiomedical Science & WCU Research Center, Dankook University, Cheonan 330-714, Republic of Korea 5Department of Pharmacy, College of Pharmacy, Gachon University of Medicine and Science, Incheon 406-799, Republic of Korea

(Received 21 May 2011, accepted 13 August 2011)

DYX1C1 is a candidate gene for developmental dyslexia and has three alternative pre-mRNA spliced forms in the human genome. One of the transcripts contains an HERV-H LTR that could affect the expression level of DYX1C1. We speculate that the HERV-H LTR integrated into the DYX1C1 locus in the catarrhine lineage after its divergence from the platyrrhine lineage. Reverse transcription-PCR of the HERV-H LTR-related transcript produced four alternative forms from several human tissues. All of alternative forms were also identified in various rhesus macaque tissues. Through sequencing analysis of various primate DNA samples, we found that a part of the HERV-H LTR sequence was duplicated within the DYX1C1 exon 9 only in catarrhines. However, the duplication event did not cause frameshift mutation of the DYX1C1 transcript. Taken together, this HERV-H LTR insertion into DYX1C1 has contributed to transcript diversification of DYX1C1 during primate evolution.

Key words: alternative splicing, duplication, DYX1C1 gene, exonization, HERV-H

2004; Kim et al., 2004). In addition, the majority of INTRODUCTION HERVs exist as solitary LTR elements resulting from Human endogenous retroviruses (HERVs) are abun- deletions associated with the homologous recombination dantly interspersed in primate genomes including the between LTRs of full-length HERVs (Hughes and Coffin, human genome (Chang et al., 2007). HERV families 2005). Some HERV subfamilies retain functional open were derived from exogenous retroviruses that infected reading frames (ORFs), mainly derived from the env the host’s germline cells (Llorens et al., 2009; Lower et al., genes of the HERV-K, HERV-H, and HERV-W subfami- 1996). Most of the HERV families in primate genomes lies (Wentzensen et al., 2007). In addition, it has been emerged after the divergence of the catarrhine and reported that HERV-LTRs are able to influence and reg- platyrrhine lineages (Huh et al., 2006a, 2006b; Sin et al., ulate the expression of adjacent genes via their strong 2006, 2007). The full-length HERV sequence is similar promoter and enhancer activities (Jern and Coffin, 2008; to a proviral DNA sequence in that it contains gag, pol, Medstrand et al., 2005; Yi et al., 2004). It has also been and env genes and is flanked by insertional long terminal demonstrated that the expression and promoter activities repeats (LTRs) of several hundred nucleotides including of HERV-H LTRs are specific to cell type (Schon et al., promoter and enhancer elements (Goodchild et al., 1995; 2001). O’Connell and Cohen, 1984). However, most of the Recent studies of the HERV integration have explored HERVs in the human genome are defective, either due to the relationships between human disease and HERVs point mutations or structural insufficiencies (e.g., trunca- (Conrad et al., 1994; Depil et al., 2002; Nelson et al., 1999; tions, deletions, and insertions) (Bannert and Kurth, Wentzensen et al., 2007). For instance, an HERV-K env transcript with protein-coding potential was identified in Edited by Hidenori Nishihara human breast cancer (Wang-Johanning et al., 2001, 2003) * Corresponding author. E-mail: [email protected] and the expression of HERV-K env protein was elevated 278 Y.-J. KIM et al. in ovarian cancers (Wang-Johanning et al., 2007). Preparing for DNA and RNA Using the Trizol reagent The HERV-H family is one of the largest and most (Invitrogen), we extracted total RNAs from the adrenal abundant HERV families in the human genome (Mager gland, cerebellum, heart, kidney, liver, lung, testis, and and Freeman, 1995; Yi et al., 2006). Among them, 100 trachea cells of rhesus macaques and the brain, heart, copies of HERV-H elements are full-length, 800–900 cop- kidney, liver, lung, and ovary cells of marmosets. The ies of the HERV-H family have no pol and env genes, and total RNAs of human tissues including adrenal gland, cer- the others (~1000 copies) exist as solitary LTRs (Hirose et ebellum, heart, kidney, liver, lung, testis, and trachea al., 1993; Yi et al., 2006). The HERV-H elements first were purchased from Clontech. We isolated mRNAs appear to have inserted into primate genomes over 40 from the total RNAs described above using PolyAtract million years ago and experienced a period of rapid mRNA Isolation systems (Promega). expansion 30–35 million years ago (Anderssen et al., According to a standard protocol (Sambrook et al., 1997; Mager and Freeman, 1995; Yi et al., 2006). An 1989), genomic DNAs were isolated from the heparinized HERV-H element located within a gene could potentially blood samples of the following species: (1) hominoids: generate alternative transcripts of the gene by providing common chimpanzee (Pan troglodytes), gorilla (Gorilla promoter activity, a poly-A signal, or alternative splicing gorilla), orangutan (Pongo pygmaeus), and a gibbon sites (Sin et al., 2007). (Hylobates agilis); (2) Old World monkeys: Japanese mon- DYX1C1 is a candidate gene of the learning disorder, key (Macaca fuscata) and rhesus macaque (Macaca dyslexia. Disruption of the DYX1C1 gene caused by a mulatta); (3) New World monkeys (Platyrrhini): spider chromosomal translocation was detected in dyslexia monkey (Ateles belzebuth), night monkey (Aotus trivirgatus), patients (McGrath et al., 2006; Taipale et al., 2003). The squirrel monkey (Saimiri sciureus), and common marmo- DYX1C1 gene is located on chromosome 15q21 and con- set (Callithrix jacchus); and (4) prosimian: ring-tailed sists of nine exons dispersed over 78 kb of genomic lemur (Lemur catta). DNA. The protein encoded by DYX1C1 is composed of 420 amino acids. In this study, we investigated a hybrid PCR and reverse transcription (RT)-PCR amplifi- transcript of DYX1C1 that contains the LTR of an HERV- cation DYX1C1 gene transcripts were analyzed by RT- H element in its last exon. The solitary LTR could poten- PCR amplification. M-MLV reverse transcriptase tially cause pretermination of the DYX1C1 transcript. A (Promega) with an annealing temperature of 42°C and an comparative analysis of DYX1C1 expression patterns in RNase inhibitor (Promega) were used for the reverse primates indicated that this solitary LTR of the HERV-H transcriptions of the transcripts. The DYX1C1 tran- element could generate lineage-specific variation in scripts related to HERV-H LTR were amplified with a DYX1C1 expression. Therefore, we suggest that the primer pair from GenBank (accession no. NM001033560.1) insertion of HERVs into genic regions may have contrib- consisting of S1 (5’-AGGCACGAAGAGCAATGAAT-3’) uted to gene-expression diversification. and AS2 (5’-GTGGGGCCGTTTTATAGGAT-3’). Two dif- ferent DYX1C1 transcripts which are not related to the HERV-H LTR were amplified with a primer pair from MATERIALS AND METHODS GenBank (accession no. NM001033559.1 and NM13081.2) Computational analysis To identify the sequences consisting of S2 (5’-GACATAGCTGAACTTTGCGATTT- related to the HERV-H LTR, we scanned the human ref- 3’) and AS2 (5’-GATTTATAATATTTTGCCCTCAACAG- erence genome sequence, human expressed sequence tags 3’). Each PCR was performed in triplicate. The PCR (ESTs), and human RefSeq mRNAs. Using the HERV-H samples were subjected to an initial denaturation step of consensus sequence as a query, we screened non-redun- 4 min at 94°C, followed by 30 cycles of PCR at 40 sec of dant databases through BLAST version 2.2.9 to identify denaturation at 95°C, 40 sec of annealing at 55°C, and 90 the novel hybrid transcripts of HERV-H and DYX1C1 sec of extension at 72°C, followed by a final extension step (Altschul et al., 1997). Then, we aligned the transcripts of 7 min at 72°C. As a standard control, the G3PDH with the DYX1C1 gene to identify the precise splicing gene was amplified with a primer pair consisting of patterns. Repeatititve elements included in the DYX1C1 GPH-S (5’-GAGCCCCAGCCTTCTCCATG-3’) and GPH-AS transcripts and their genomic loci were identified using (5’-GAAATCCCATCACCATCTTCCAGG-3’) from human the RepeatMasker program (www.repeatmasker.org) ref- G3PDH (GenBank accession no. NM002046). erencing a library containing the consensus sequences from Repbase Update (Jurka, 2000). Using the EST and RESULTS AND DISCUSSION RefSeq mRNAs, we accurately reconstructed the gene structure of DYX1C1. To analyze the sequences of the Identification and characterization of DYX1C1 various DYX1C1 transcripts, we aligned them using the hybrid transcripts HERVs are among the most stud- ClustalW program (Thompson et al., 1994). ied transposable elements in the human genome and have the potential to regulate the expression of genes by pro- DYX1C1 transcripts diversification by HERV-H LTR 279 viding promoter activity or poly-A signals. Three alter- Expression analysis of alternatively spliced DYX1C1 native splicing forms of the DYX1C1 gene were identified transcripts in human, rhesus macaque, and marmo- from the GenBank database. Among them, one tran- set The coding sequence of the DYX1C1 gene contains an script contains a single LTR region of an HERV-H ele- HERV-H LTR element. To confirm whether the LTR ment (NM001033560) but the others include no HERV- sequences are expressed, we conducted RT-PCR with sev- related sequences (Fig. 1). The HERV-H LTR is inserted eral human tissues. It was previously reported that the in the sense-orientation into the last exon of the DYX1C1 alternatively spliced DYX1C1 transcripts are ubiquitously gene, providing the gene with additional protein-coding expressed in several human tissues. As can be seen in sequences and a canonical poly-A signal (AATAA). It Fig. 2, four alternative spliced forms were identified in has been previously reported that the solitary LTR of an human tissues through the RT-PCR approach. The four HERV-H element integrated near a gene may lead to var- alternative spliced forms (V1-1, V1-2, V1-3, and V1-4) ious transcripts, some of which contain the LTR. resulted in DYX1C1 either acquiring novel exons or losing NADSYN1 (NAD synthetase) is a well-known example. original exons. The PCR products of V1-1 and V1-2 It has several alternative transcripts resulting from showed only a subtle difference in size, though V1-1 con- HERV-H LTR-derived poly-A signal and these transcripts tains an extra exon compared to V1-2 (Kim et al., 2009). have been detected in various normal tissues and cancer We also confirmed these alternative forms by sequencing cells (Sin et al., 2007). In addition, a recent study about them. exonization of LTR transposable elements in the human In this study, we found that all five transcripts are genome showed that 1,057 human genes are associated ubiquitously expressed in human tissues, although the with LTR retrotransposons (Piriyapongsa et al., 2007). original transcript of the DYX1C1 gene is expressed more Our study provides increasing evidence that LTR ret- strongly than the alternative transcripts. This result is rotransposons have played an important role in creating concordant with a previous report on the expression of the gene diversity, such as hybrid transcripts, in the human DYX1C1 gene (Kim et al., 2009). DYX1C1 has a role in genome. the migration of neocortical neurons and, more specifi-

Fig. 1. Structure of the DYX1C1 gene and its alternatively spliced variants. The DYX1C1 gene is located on chromosome 15q21.3. Three alternative spliced transcripts, V1, V2, and V3, from the GenBank database are illustrated. V1 is associated with a solitary HERV-H LTR insertion in exon 9 (arrow) while V2 and V3 lack this insertion. 280 Y.-J. KIM et al.

Fig. 2. RT-PCR analysis of DYX1C1 transcripts between human, rhesus macaque, and marmoset tissues. Three transcripts are expressed in all assayed human and rhesus macaque tissues. Two arrows (S, forward primer; AS, reverse primer) indicate the posi- tions at which each oligonucleotide primer anneals during RT-PCR. Tissues assayed in human and rhesus macaque: Lane 1, adrenal gland; Lane 2, cerebellum; Lane 3, heart; Lane 4, kidney; Lane 5, liver; Lane 6, lung; Lane 7, testis; Lane 8, trachea. In marmoset tissues, V2 and V3, which do not contain the HERV-H LTR element, are expressed. Tissues assayed in marmoset: Lane 1, brain; Lane 2, heart; Lane 3, kidney; Lane 4, liver; Lane 5, lung; Lane 6, ovary. G3PDH was amplified as a positive control in all instances. cally, is required during the transition out of the multipo- came to exist within the DYX1C1 gene. One possible lar stage of migration (Wang et al., 2006). For these mechanism involves homologous recombination between reasons, we conducted RT-PCR with several parts of the the two LTRs of the HERV-H after its insertion into the human brain. The results show that DYX1C1 is broadly host genome in exon 9 of the DYX1C1 gene, resulting in expressed throughout the human brain. In addition, we a single remaining LTR at the locus. Then, this solitary performed RT-PCR with various rhesus macaque tissues LTR allele became fixed in the ancestral catarrhine pop- and detected five DYX1C1 transcripts, which are identical ulation. to those observed in human tissues (Fig. 2). However, HERVs have the potential to modify the expression of RT-PCR using marmoset tissues showed that the expres- neighboring genes. Most directly, HERV insertion into a sion pattern of the marmoset DYX1C1 differs from those gene could change the amino acid sequence of the gene, of human and rhesus macaque DYX1C1; the transcript causing tissue- or disease-specific expression. Addition- containing an HERV-H LTR (V1) was not expressed, ally, HERV insertions can modify the expression of while the other two transcripts (V2 and V3) were detected nearby genes by altering promoter activity, providing in marmoset tissues. The standard RT-PCR showed poly-A signals, or donating alternative splice sites. One similar expression levels of V2 and V3 transcripts in interesting example, the HERV-K element, has full- human tissues. However, we found that the transcrip- length intact ORFs, and their remnants and proteins tional activity of V3 was higher than that of V2 in tumor have been detected in breast cancer (Frank et al., 2008; tissue than in normal tissue using semi-quantitative RT- Golan et al., 2008; Wang-Johanning et al., 2008), and tes- PCR analysis in a previous report (Kim et al., 2009). tis cancer (Goedert et al., 1999; Ruprecht et al., 2008). Based on these results, we suggest that the HERV-H LTR Our data adds to the mounting evidence of HERV-associ- element was inserted into the catarrhine lineage after its ated gene expression alteration by showing that an divergence from the platyrrhine lineage. Several HERV-H LTR insertion affects the expression of the hypotheses may explain how a solitary HERV-H LTR DYX1C1 gene by providing a poly-A signal. These obser- DYX1C1 transcripts diversification by HERV-H LTR 281 vations, combined with those from previous research, pro- sequences and aligned the translated amino acid vide evidence that HERVs may alter critical biological sequences. Through this process, we found that the functions and affect oncogenic process leading to the HERV-H LTR preterminates the translation of DYX1C1 development of specific human tumors. by providing a stop codon. Thus, the length of the mRNA became shorter during catarrhine evolution. Evolutionary aspect of the hybrid transcript of As shown in Fig. 4, although a part of the HERV-H LTR the DYX1C1 gene To investigate the evolutionary his- sequence within exon 9 of the DYX1C1 gene may be either tory of the HERV-H LTR in primate genomes, we per- duplicated in the Old World monkey lineage or deleted in formed PCR amplifications using genomic DNAs from the hominoid, the genomic alteration did not cause frame- twelve primates as templates. As shown in Fig. 3, the shift mutation of the DYX1C1 transcript. To determine expected PCR products of 799 bp containing the HERV-H whether the 39-bp difference was generated by a duplica- LTR were recovered from the catarrhines (hominoids and tion or deletion event, we tried to find another copy of the Old World monkeys) but not from the platyrrhines or 39-bp sequence in primate genomes using the BLAST-Like prosimian. It indicates that HERV-H element inte- Alignment Tool utility (http://genome.ucsc.edu/cgi-bin/ grated into primate genomes after the divergence of the hgBlat). We reasoned that if the extra 39-bp existed Catarrhini and Platyrrhini lineages. It was reported prior to the divergence of the hominoid and Old World that the DYX1C1 protein sequence of non-human pri- monkey lineages, then we should be able to find other mates is 98.6–99.5% similar to that of human (Taipale et HERV copies containing the extra 39-bp in the available al., 2003). To examine DYX1C1 protein sequences catarrhine genomes. However, we could not find any among primates, we sequenced the 799 bp PCR products. additional HERV elements with the extra 39-bp sequence We then conducted putative translations of the DNA in human, chimpanzee, orangutan, or rhesus macaque.

Fig. 3. Phylogenetic extent and orthologous structure of the solitary HERV-H LTR insertion locus in primate lineages. (A) Gel chro- matographs showing PCR amplification of the HERV-H LTR-containing portion of exon 9 of DYX1C1 in twelve primates. Species are abbreviated as follows: HU, human; CH, chimpanzee; GO, gorilla; OR, orangutan; GI, gibbon; JM, Japanese macaque; RH, rhesus macaque; SM, spider monkey; NM, night monkey; CM, common marmoset; SQ, squirrel monkey; and RT, ring-tailed lemur. M indicates a size marker. (B) Alignment of HERV-H LTR-containing sequences in exon 9 of the V1 transcript. The thick black horizontal line represents the 9th exon sequence and the grey box indicates the HERV-H LTR element insertion, which is only found in catarrhines. The solid box indicates a 39-bp duplicated sequence present only in Old World monkeys. 282 Y.-J. KIM et al.

Fig. 4. Amino acid alignment of LTR-derived sequences in the protein-coding region of human and five other catarrhines. The gray box indicates duplicated sequences present only in Old World monkeys. The solid box shows the stop codons produced by the HERV- H LTR sequence in each species. CDS, coding DNA sequence. Species are abbreviated as follows: HU, human; CH, chimpanzee; GO, gorilla; GI, gibbon; JM, Japanese macaque; and RH, rhesus macaque.

Therefore, we conclude that the extra 39-bp sequence was We thank Drs. Jungnam Lee and Joshua Meyer for comments likely generated by an Old World monkey lineage-specific on the manuscript. This research was supported by the Bio- duplication event. Scientific Research Grant funded by the Pusan National University (PNU-2008-101-20080596000) and WCU (World Class University) We further investigated the orthologous loci of the program through the National Research Foundation of Korea DYX1C1 gene containing the HERV-H LTR in the mouse funded by the Ministry of Education, Science and Technology and rat genomes. Mouse SINEs (Short INterspersed (R31-10069). Elements) of the PB1D9 and B1F subfamilies were identified in the rodent DYX1C1 genes, indicating that they REFERENCES were inserted into the rodent genome after the divergence of the rodent and primate lineages (data not shown) (Ding Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and et al., 2005; Ponicsan et al., 2010). It has been suggested PSI-BLAST: a new generation of protein database search that the HERV-H subfamily emerged before the diver- programs. Nucleic Acids Res. 25, 3389–3402. gence of platyrrhine and catarrhine lineages, ~40 million Anderssen, S., Sjøttem, E., Svineng, G., and Johansen, T. (1997) years ago. Amplification rates of HERV-H remained rel- Comparative analyses of LTRs of the ERV-H family of pri- atively low in the platyrrhines but appears to have under- mate-specific retrovirus-like elements isolated from marmoset, African green monkey, and man. Virology 234, 14–30. gone rapid expansion in catarrhine genomes prior to the Bannert, N., and Kurth, R. (2004) Retroelements and the human divergence between Old World monkeys and hominoids genome: new perspectives on an old relation. Proc. Natl. (Mager and Freeman, 1995). Acad. Sci. USA 101, 14572–14579. In conclusion, we suggest that the HERV-H LTR Chang, N. T., Yang, W. K., Huang, H. C., Yeh, K. W., and Wu, sequence has played a biological role in the evolution of the C. W. (2007) The transcriptional activity of HERV-I LTR is negatively regulated by its cis-elements and wild type p53 DYX1C1 gene during the catarrhine radiation by providing tumor suppressor protein. J. Biomed. Sci. 14, 211–222. additional protein-coding regions and a poly-A Conrad, B., Weidmann, E., Trucco, G., Rudert, W. A., Behboo, signal. Although further analyses are required to eluci- R., Ricordi, C., Rodriquez-Rilo, H., Finegold, D., and Trucco, date conclusively the functions of the transcriptional vari- M. (1994) Evidence for superantigen involvement in insulin- ants of the DYX1C1 gene in primate genomes, the results dependent diabetes mellitus aetiology. Nature 371, 351– 355. of this study will help us understand the characteristics of Depil, S., Roche, C., Dussart, P., and Prin, L. (2002) Expression the HERV-H LTR insertion driving the transcript diversi- of a human endogenous retrovirus, HERV-K, in the blood fication and its effect on the evolution of DYX1C1 gene. cells of leukemia patients. Leukemia 16, 254–259. DYX1C1 transcripts diversification by HERV-H LTR 283

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