Genome-wide analysis of DNA methylation patterns in horse.

BACKGROUND: DNA methylation is an epigenetic regulatory mechanism that plays an essential role in mediating biological processes and determining phenotypic plasticity in organisms. Although the horse reference genome and whole transcriptome data are publically available the global DNA methylation data are yet to be known. RESULTS: We report the first genome-wide DNA methylation characteristics data from skeletal muscle, heart, lung, and cerebrum tissues of thoroughbred (TH) and Jeju (JH) horses, an indigenous Korea breed, respectively by methyl-DNA immunoprecipitation sequencing. The analysis of the DNA methylation patterns indicated that the average methylation density was the lowest in the promoter region, while the density in the coding DNA sequence region was the highest. Among repeat elements, a relatively high density of methylation was observed in long interspersed nuclear elements compared to short interspersed nuclear elements or long terminal repeat elements. We also successfully identified differential methylated regions through a comparative analysis of corresponding tissues from TH and JH, indicating that the gene body regions showed a high methylation density. CONCLUSIONS: We provide report the first DNA methylation landscape and differentially methylated genomic regions (DMRs) of thoroughbred and Jeju horses, providing comprehensive DMRs maps of the DNA methylome. These data are invaluable resource to better understanding of epigenetics in the horse providing information for the further biological function analyses.

Transcriptional variations mediated by an alternative promoter of the FPR3 gene.

Formyl peptide receptor 3 (FPR3) is a potential player in innate immunity and appears with FPR2 as a FPR cluster during primate evolution. Comparative genome analyses indicate that a segmental duplication (SD) event upstream of the FPR3 gene after the divergence of New and Old World monkeys led to the emergence of an alternative promoter. In this study we combined computational and experimental approaches to identify a FPR3 gene that is controlled by an alternative promoter derived during a SD event. Its transcriptional activity was detected by quantitative reverse transcription polymerase chain reaction. Human alternative transcripts (FPR3-1 and FPR3-2) showed tissue-specific patterns with strong expressions in lung or uterus, while the FPR3-1 transcript of rhesus macaque is broadly expressed in various tissues. Overall, transcriptional variations of FPR3 occur by an alternative promoter during primate evolution.

Quantitative analysis of alternative transcripts of human PCDH11X/Y genes.

The Protocadherin 11X/Y (PCDH11X/Y) gene pair has been proposed as a carrier of the variation relating to cerebral asymmetry and psychosis on the ground that the Y gene was generated by duplication at 6 million years (close to the chimpanzee-human separation) and there is a case for an X/Y determinant of cerebral asymmetry. The present article investigated the patterns of alternative splicing and expression of the PCDH11X/Y genes. Twelve alternative transcripts of PCDH11X/Y genes were presently identified by in silico analysis. To investigate the biological roles of alternative transcripts of PCDH11X/Y genes, the transcripts were analyzed by real-time reverse transcription-polymerase chain reaction amplification. A total of 31 normal tissues including 11 different regions of human brain were used to investigate a wide spectrum of expression profiles. Dominant expression patterns were identified in several tissues (Tx1-fetal liver; Tx3-adult brain; Tx4-adult brain and kidney; Tx5-bone marrow; Ty1-fetal brain; Ty2-adrenal gland). Tx4 transcripts showed specific expression patterns in olfactory tissues. The findings can guide functional investigation of neuropsychiatric disorders.

Identification and molecular characterization of PERV gamma1 long terminal repeats.

Porcine endogenous retroviruses (PERVs) gamma1 in the pig genome have the potential to act as harmful factors in xenotransplantation (pig-to-human). Long terminal repeats (LTRs) are known to be strong promoter elements that could control the transcription activity of PERV elements and the adjacent functional genes. To investigate the transcribed PERV gamma1 LTR elements in pig tissues, bioin-formatic and experimental approaches were conducted. Using RT-PCR amplification and sequencing approaches, 69 different transcribed LTR elements were identified. And 69 LTR elements could be divided into six groups (15 subgroups) by internal variation including tandem repeated sequences, insertion and deletion (INDEL). Remarkably, all internal variations were indentified in U3 region of LTR elements. Taken together, the identification and characterization of various PERV LTR transcripts allow us to extend our knowledge of PERV and its transcriptional study.

Selection of internal reference genes for SYBR green qRT-PCR studies of rhesus monkey (Macaca mulatta) tissues.

BACKGROUND: The rhesus monkey (Macaca mulatta) is a valuable and widely used model animal for biomedical research. However, quantitative analyses of rhesus gene expression profiles under diverse experimental conditions are limited by a shortage of suitable internal controls for the normalization of mRNA levels. In this study, we used a systematic approach for the selection of potential reference genes in the rhesus monkey and compared their suitability to that of the corresponding genes in humans. RESULTS: Eight housekeeping genes (HKGs) (GAPDH, SDHA, ACTB, RPL13A, RPL32, UBA52, PGK1Y, and YWHAZ) from rhesus monkeys and humans were selected to test for normalization of expression levels in six different tissue types (brain, colon, kidney, liver, lung, and stomach). Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. Intriguingly, RPL13A and RPL32 were selected as ideal reference genes only in rhesus monkeys. CONCLUSION: The results clearly indicated the necessity of using different reference genes for normalization of expression levels between rhesus monkeys and humans in various tissues.

Cooperative exonization of MaLR and AluJo elements contributed an alternative promoter and novel splice variants of RNF19.

The RNF19 protein, which contains RING-finger and IBR motifs, acts as an E3 ubiquitin ligase localized to Lewy bodies. RNF19 is located on human chromosome 8q22.2, has a 4.4-kb transcript, and is ubiquitously expressed in various tissues. Here, we identified an alternative RNF19 promoter region and alternative RNF19 transcripts derived from MaLR (mammalian apparent LTR-retrotransposon) and AluJo elements. Comparative analyses indicated human-specific expression of the MaLR- and AluJo-related transcripts. From the expression analysis of 72 tissue samples including human normal, tumor, and primate tissues, three different isoforms (V1, V2, and V3) of MaLR-derived transcripts were identified. Quantitative RT-PCR analysis showed a dominant expression pattern of the V2 MaLR-derived transcript. A reporter gene assay for MaLR element promoter activity indicated that pGL2-RNF19/MaLR in the forward orientation is capable of driving luciferase gene expression in Cos7 and HCT116 cells. These findings suggest that RNF19 has acquired a new promoter and alternative exons via continuous retrotransposition events of MaLR and AluJo elements during mammalian and primate evolution, respectively.

Promoter activity of the long terminal repeats of porcine endogenous retroviruses of the Korean domestic pig.

Porcine endogenous retroviruses (PERVs) in the pig genome represent a potential risk of infection in pig-to-human transplantation and are transmitted vertically. The solitary long terminal repeat (LTR) elements of the PERVs affect the replication properties of the individual viruses via their repeat sequences and by encoding a set of specific transcription factors. We examined the promoter activities of solitary LTR elements belonging to the PERV-A and -B families of the Korean domestic pig (KDP) using luciferase reporters. Three of the LTR structures (of PERV-A5-KDP, PERV-A7-KDP, PERV-A8-KDP) had different promoter activities in human HCT116 cells and monkey Cos7 cells, and potential negatively and positively acting regions affecting transcription were identified by deletion analysis. These data suggest that specific sequences in the U3 region of a given LTR element can affect the activities of promoter or enhancer elements in the PERV.

Formation of a new solo-LTR of the human endogenous retrovirus H family in human chromosome 21.

Human endogenous retroviruses (HERVs) contribute to various kinds of genomic instability via rearrangement and retrotransposition events. In the present study the formation of a new human-specific solo-LTR belonging to the HERV-H family (AP001667; chromosome 21q21) was detected by a comparative analysis of human chromosome 21 and chimpanzee chromosome 22. The solo-LTR was formed as a result of an equal homologous recombination excision event. Several evolutionary processes have occurred at this locus during primate evolution, indicating that mammalian-wide interspersed repeat (MIR) and full-length HERV-H elements integrated into hominoid genomes after the divergence of Old World monkeys and hominoids, and that the solo-LTR element was created by recombination excision of the HERV-H only in the human genome.

Endogenous retrovirus-related sequences provide an alternative transcript of MCJ genes in human tissues and cancer cells.

The MCJ gene is a member of the DNAJ family, and its transcriptional event is controlled by methylation of the CpG island. In our study, we found LTR33 and LTR7 elements provided an alternative transcript within the MCJ gene. To detect different expression patterns between the originally reported MCJ transcript and the LTR-related transcript, we performed a RT-PCR approach using various human tissues and cancer cells. The original MCJ transcript was detected in human tissues and cancer cells, whereas the LTR-related transcript was only revealed in some cancer cells (HCT106, MCF-3, TE-1, Hela, and CCHM). We also performed a PCR analysis to compare the insertion lineage of the LTR elements with the genomic DNAs of primates, indicating that those LTR33 and LTR7 elements of HERV-H have been integrated into the primate genome at different times. Taken together, we suggest that HERV-related elements trigger transcriptome diversification during primate evolution.

Genome-wide analysis of DNA methylation before-and after exercise in the thoroughbred horse with MeDIP-Seq.

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.

Genome-Wide Identification and Classification of MicroRNAs Derived from Repetitive Elements.

MicroRNAs (miRNAs) are known for their role in mRNA silencing via interference pathways. Repetitive elements (REs) share several characteristics with endogenous precursor miRNAs. In this study, 406 previously identified and 1,494 novel RE-derived miRNAs were sorted from the GENCODE v.19 database using the RepeatMasker program. They were divided into six major types, based on their genomic structure. More novel RE-derived miRNAs were confirmed than identified as RE-derived miRNAs. In conclusion, many miRNAs have not yet been identified, most of which are derived from REs.

The novel MER transposon-derived miRNAs in human genome.

MicroRNAs (miRNAs) are small RNA molecules (~20-30 nucleotides) that generally act in gene silencing and translational repression through the RNA interference pathway. They generally originate from intergenic genomic regions, but some are found in genomic regions that have been characterized such as introns, exons, and transposable elements (TE). To identify the miRNAs that are derived from palindromic MERs, we analyzed MER paralogs in human genome. The structures of the palindromic MERs were similar to the hairpin structure of miRNA in humans. Three miRNAs derived from MER96 located on chromosome 3, and MER91C paralogs located on chromosome 8 and chromosome 17 were identified in HeLa, HCT116, and HEK293 cell lines. The interactions between these MER-derived miRNAs and AGO1, AGO2, and AGO3 proteins were validated by immunoprecipitation assays. The data suggest that miRNAs derived from transposable elements could widely affect various target genes in the human genome.

Identification and promoter analysis of PERV LTR subtypes in NIH-miniature pig.

Porcine endogenous retroviruses (PERVs) are integrated into the genomes of all pigs. Since some PERVs can also infect human cells, they represent a potential risk for xenotransplantation involving pig cells or organs. The long terminal repeat (LTR) elements of PERVs show promoter activity that can affect human functional genes; therefore, we examined these elements in this study. We detected several expressed LTRs in the NIH-miniature pig liver, among which we identified 9 different subtypes. When these LTRs were compared, distinct structures that contained several insertion and deletion (INDEL) events and tandem repeats were identified in the U3 region. The transcriptional activity of the 9 LTR subtypes was analyzed in the PK15 porcine cell line and in the HepG2 and Hep3B human liver cell lines, and transcriptional regulation was found to be different in the 3 cell lines. The D LTR subtype was found to have stronger promoter activity than all other types in 4 different human cell lines (HepG2, Hep3B, U251, and 293). Using computational approaches, the D type was shown to contain 4 transcription factor-binding sites distinct from those in the U3 regions of the other subtypes. Therefore, deletion mutants were constructed and examined by a transient transfection luciferase assay. The results of this analysis indicated that the binding site for the Hand1:E47 transcription factor might play a positive role in the transcriptional regulation of PERV LTR subtype D in human liver cell lines.

Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig.

The potential risk of porcine endogenous retrovirus (PERV) transmission is an important issue in xenotransplantation (pig-to-human transplantation). Long terminal repeats (LTRs) in PERV elements show promoter activity that could affect neighboring functional genes. The methylation status and promoter activities of 3 LTR structures (PERV-LTR1, LTR2, and LTR3 elements) belonging to the PERV-A family were examined using luciferase reporter genes in human liver cell lines (HepG2 and Hep3B). The PERV LTR3 element exhibited hypomethylation and stronger promoter activity than the other LTR elements in human liver cells. We also performed comparative sequences analysis of the PERV LTR elements by using bioinformatics tools. Our findings showed that several transcription factors such as Nkx2-2 and Elk-1 positively influenced the high transcriptional activity of the PERV LTR3 element.

Quantitative analysis of transcript variants of CHM gene containing LTR12C element in humans.

Choroideremia (CHM) is essential for the posttranslational activation of retina-specific Rab protein. Transcript variants (a and b) of the CHM gene were detected in human cancer cells and tissues. Sequence analysis of the both variants found that isoform b is caused by an LTR12C element offering an alternative splicing site within the CHM gene. We performed real-time RT-PCR analysis to study expression levels of the CHM transcript variants in tumor and adjacent normal tissue samples. Our results showed that CHM isoform b was highly expressed in tumor tissues but its expression levels were relatively low in those of adjacent normal tissues including colon, testis, and lung. In addition, high expression levels of CHM isoform b were detected in the cancer cell lines of colon and lung, and colon cancer patient tissues. Thus, we suggest that expression levels of alternative transcripts of the CHM gene could be used as a molecular marker system to identify human cancers.

Analysis of the molecular and regulatory properties of active porcine endogenous retrovirus gamma-1 long terminal repeats in kidney tissues of the NIH-Miniature pig.

The pig genome contains the gamma 1 family of porcine endogenous retroviruses (PERVs), which are a major obstacle to the development of successful xenotransplantation from pig to human. Long terminal repeats (LTRs) found in PERVs are known to be essential elements for the control of the transcriptional activity of single virus by different transcription factors (TFs). To identify transcribed PERV LTR elements, RT-PCR and DNA sequencing analyses were performed. Twenty-nine actively transcribed LTR elements were identified in the kidney tissues of the NIH-Miniature pig. These elements were divided into two major groups (I and II), and four minor groups (I-1, I-2, I-3, and II-1), by the presence of insertion and deletion (INDEL) sequences. Group I elements showed strong transcriptional activity compared to group II elements. Four different LTR elements (PL1, PL2, PL3, and PL4) as representative of the groups were analyzed by using a transient transfection assay. The regulation of their promoter activity was investigated by treatment with M.SssI (CpG DNA methyltransferase) and garcinol (histone acetyltransferase inhibitor). The transcriptional activity of PERV LTR elements was significantly reduced by treatment with M.SssI. These data indicate that transcribed PERV LTR elements harbor sufficient promoter activity to regulate the transcription of a single virus, and the transcriptional activity of PERV LTRs may be controlled by DNA methylation events.

Lineage specific evolutionary events on SFTPB gene: Alu recombination-mediated deletion (ARMD), exonization, and alternative splicing events.

SFTPB gene encoding pulmonary surfactant protein has been investigated to have transcript variants related with transposable elements (TEs). To investigate the alternative splicing event on SFTPB gene, various primate samples (tissue RNA and genomic DNA) were used. Two different transcript variants (T1-2 and T2-2) were newly identified by RT-PCR analysis. T1-1,-2 and T2-1,-2 are TE-related and original transcripts, respectively. T1-1 transcript was investigated to be a lung and human specific alternative transcript. T2-1 transcript shows dominant expression pattern in lung tissues. T1-related TEs (LTR7B and AluSx) were investigated by genomic PCR and sequencing procedure of different primate genomic DNAs. Sequencing analysis indicated that they had been integrated into our common ancestor genome before the divergence of simian and prosimian and Alu recombination-mediated deletion (ARMD) event had been occurred on our common ancestor genome after the divergence of New World monkeys and Old World monkeys in SFTPB gene 3' UTR region. Taken together, integration event of LTR7B and AluSx on SFTPB gene seem to cause lineage specific events via general exonization event (New World monkeys and prosimian), ARMD event (Old World monkeys and hominoids), and alternative splicing (human) during the primate evolution.

Molecular characterization of the DYX1C1 gene and its application as a cancer biomarker.

PURPOSE: DYX1C1 has three alternatively spliced transcripts. Therefore, we expect that alternative transcripts of DYX1C1 are used as a biomarker to detect specific cancer. METHODS: RT-PCR analysis is conducted in order to detect expression of the DYX1C1 gene and the PCR products were analyzed using the Image J program to compare the expression levels of each transcript. RESULTS: We found one of the transcripts was directly associated with an HERV-H LTR element that could be translated into protein sequence. Four new alternative transcripts were identified by RT-PCR analysis with various human tissue samples including 10 normal and adjacent tumor tissue sets. Semi-quantitative RT-PCR analysis showed the transcriptional activity of V3 and V2 was higher in tumor than in normal tissue samples, especially in the colorectal tissue samples. CONCLUSION: Our results indicated that alternatively spliced transcript variants of the DYX1C1 gene could be used as cancer biomarkers to detect colorectal cancer.