H.-S. Kim

Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells

Summary.Long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) have been reported to serve as alternative promoters in functional genes. The GSDML (gasdermin-like protein) gene located on human chromosome 17q21 has been found to be an oncogenomic recombination hotspot. Here, we identified the LTR element of HERV-H with reverse orientation as an alternative promoter of the GSDML gene and analyzed its expression pattern in human tissues and cancer cells. A reporter gene assay of the promoter activity of the LTR on the GSDML gene in human cancer cell lines (HCT-116 and HeLa) and a kidney cell line (Cos7) of African green monkey indicated that the LTR promoter with reverse orientation had stronger promoter activity than forward one. The transcripts of this LTR-derived promoter were widely distributed in various human tissues and cancer cells, whereas the transcripts of the cellular promoter were found only in stomach tissues and some cancer cells (HCT116, MCF7, U937, C-33A, and PC3). These findings suggest that the LTR element on the GSDML gene was integrated into the hominoid lineage and acquired the role of transcriptional regulation of human tissues and cancer cells.

Transcriptional regulation of GSDML gene by antisense-oriented HERV-H LTR element

During the course of hominoid evolution, a new transcript variant of the GSDML (gasdermin-like protein) gene was formed by the integration of the antisense-oriented HERV-H (human endogenous retrovirus) LTR (long terminal repeat) element. To investigate regions that are critical for transcriptional regulation of the GSDML gene, we generated seven deletion mutants from a full-length clone (clone 1/630) that includes the HERV-H LTR sequence and compared their expression levels relative to the full-length parental clone using a transient transfection assay. In the transient transfection assay, deletion of the 5′ flanking region (cellular origin) of the HERV-H LTR sequence led to a 4.5-fold increase in expression compared to the full-length clone, while deletion of the U5 region showed a significant decrease in transcriptional activity. Deletion of the 3′ flanking region of the LTR sequence (clone 42/451) showed similar transcriptional activity to a clone missing the 5′ flanking region of cellular origin (clone 42/630). Taken together, these data indicate that the HERV-H LTR sequence (viral origin) positively regulates transcriptional activity of the GSDML gene and that the 5′ flanking region sequence (cellular origin) exerts negative transcriptional regulation.

Placenta-Restricted Expression of LTR-Derived NOS3

Domestication events of long terminal repeat (LTR) sequences of the human endogenous retrovirus (HERV) family have been considered to be a new mechanism for the generation of alternative splicing in the human genome. We investigated an LTR10A belonging to the HERV-I family at the human endothelial nitric oxide synthase (NOS3) gene locus. The LTR10A element was located upstream of the original promoter sequences of NOS3. Expression analysis using RT-PCR and reporter gene assays in HCT116 and COS7 cells indicated placenta-specific expression of NOS3 driven by the LTR10A-derived promoter. The placenta-restricted expression was also determined to be associated with hypomethylation of the LTR10A element by methylation analysis using sodium bisulfite DNA sequencing. Furthermore, treatment of brain-derived cell lines with demethylation reagents did not restore expression of the LTR-derived NOS3 gene transcript. Taken together, the integration event of an LTR10A element in the upstream region of NOS3 led to the generation of a placenta-specific alternative transcript governed by cooperative mechanisms of epigenetic control (DNA methylation) and transcriptional regulation (interaction between cis- and trans-acting elements).

Quantitative expression of the HERV-W env gene in human tissues

Human endogenous retroviruses HERV-W families have been identified from monozygotic twin pairs with schizophrenia and patients with multiple sclerosis. Identification of retroviral RNA in the cerebrospinal fluids and brains of individuals with schizophrenia indicated that the transcriptional activation of the HERV-W elements within the central nervous system could be associated with the brain diseases. Here, we examined the expression of the HERV-W env gene in tumor/normal adjacent tissues and various areas of brain tissues. In silico expression data indicated that 14 complete HERV-W families from human chromosomes 1, 2, 4, 7, 8, 11, 13, 15, and Y are randomly expressed in various cancer tissues. HERV-W env transcripts did not show significant differences among the human tumor/normal adjacent tissues (colon, liver, uterus, breast, and stomach). Quantitative real-time RT-PCR analysis indicated that strong expression of the HERV-W env gene was detected in the cerebral cortex and pons of the human brain.

Molecular evolution of the HERV-E family in primates

Summary.More than 50 copies of HERV-E family elements have been estimated to exist in the human genome. Here we examined the recent evolutionary history of the HERV-E family by a PCR approach using genomic DNA from hominoid primates and a human monochromosomal panel. From the HERV-E family, 25 and 68 env fragments, were identified and analyzed from hominoid primates and human chromosomes 1, 2, 3, 4, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, X, and Y, respectively. They showed 76.7–99.6% sequence similarity to that of HERV-E (accession no. M10976). Phylogenetic analysis of HERV-E env family distinctively divided into two groups (groups I and II) that each contained three subgroups. Divergence times of the two groups were estimated as 10.7 MYr for group I and 41.3 MYr for group II using an average evolutionary rate of 0.3% per MYr. These data are consistent with that of PCR analysis, which showed a band of the HERV-E family in the genomes of the hominoids, Old World monkeys, and New World monkeys. Therefore, the HERV-E family may have integrated into the primate genome after prosimians and New World monkeys diverged. Then they proliferated extensively in the genome of humans and great apes during primate evolution.

Isolation and phylogeny of endogenous retrovirus HERV-F family in Old World monkeys

Summary.u2002A new human endogenous retroviral family (HERV-F) has recently been identified from human chromosome 7q31.1-q31.3 that was identical to the XA34 cDNA clone isolated from a human glioma cDNA library with an ERV-9 env probe. We investigated pol fragments of the HERV-F family from the Old World monkeys (crab-eating monkey, African green monkey, and baboon) and analyzed these with the HERV-F (Hu-XA34). Fifteen pol fragments of the HERV-F family were detected from the Old World monkeys. They showed a high degree of sequence similarity (81–99%) with that of the HERV-F (Hu-XA34). Phylogenetic analysis of pol fragments with those of the human genome distinctively showed five groups, indicating that HERV-F family could be amplified at least five times after the original integration into the monkey genome or represent integration events independently during primate evolution.

Long terminal repeats of porcine endogenous retroviruses in Sus scrofa

SummaryUsing PCR, sequencing, and bioinformatic approaches with the genomic DNAs of Korean pigs (domestic, wild, and hybrid with Yorkshire), twelve solitary PERV long terminal repeat elements were identified and analyzed. Structure analysis of the LTR elements indicated that they have different repeat sequences in the U3 region. The PERV-A6-KWP1 and -KWP2 elements bear seven and eight 39-bp repeats, respectively. The R region of the PERV LTR elements was highly conserved in pig and mouse genomes, suggesting that they seem to have originated from a common exogenous viral element and then evolved independently throughout the course of mammalian evolution.

Molecular cloning and phylogeny of HERV-E family that is expressed in japanese monkey (Macaca fuscata) tissues

Summary.More than 50 copies of HERV-E family have been estimated to exist in the human genome. Here, we examined the expression pattern and their relationships of the HERV-E in Japanese monkey tissues by RT-PCR and sequence analysis. The env gene of HERV-E family was expressed in monkey tissues (testis, prostate, kidney, thymus, intestine and stomach) except for cerebellum, pancreas and ovary, exhibiting that they may have transcriptional potential. Phylogenetic analysis of the HERV-E env family from Japanese monkey tissues and our previous data could be divided into two distinctive groups (I and II). They were integrated into the genomes of anthropoids and have evolved at the rate of 0.3% nucleotide differences per MYr through evolutionary divergence in primate evolution. Divergence times of the two groups were estimated as 11.6u2009MYr for group I and 41.6u2009MYr for group II. Those HERV-E sequences were extensively proliferated in the genome of humans and great apes. These data will contribute to further studies on the transcriptional potential of the HERV-E family in the Japanese monkey genome and to biomedical knowledge related to human diseases.

Comparative analysis of Y chromosomal microdeletions in Korean infertile men of 47,XXY and 46,XY karyotypes

In the azoospermic patients, there are many of undiagnosed factors related to genetic bases. Among them, Klinefelters syndrome (47,XXY; KS) and Y-chromosomal microdeletion with normal karyotype (46,XY; YMNK) are the most frequent causes of male infertility. This research focused on the comparative analysis of YMNK (n = 66) and KS (n = 30) patients suffered from male infertility in Korean population. We used the polymerase chain reaction (PCR) approach including 19 pairs of sequencetagged site (STS) primers for detecting the Y-chromosomal microdeletion on AZFa, b, c regions, indicating that Y chromosomal microdeletions were almost evenly occurred in AZF all regions in Korean population. Comparative analysis indicated that 34.9% YMNK and 73.4% KS patients harbored the microdeleted Y-chromosome. It seems to be high instability of Y-chromosome in KS patients than that of YMNK infertility patients. Taken together, genome instability containing microdeletion could bring male infertility with the disturbance of normal spermatogenesis.

Expression analysis of RBMY1, CDY1, and VCY2 genes in Korean male infertility

Azoospermia factor (AZFa, b, and c) regions have been focused on their involvement in the spermatogenic process by frequent observation of microdeletion in male infertility. Among the azoospermia factors, RBMY1, CDY1, and VCY2 genes are strongly associated with the male germinal cell differentiation and development in testis. Using RT-PCR approach, expression patterns of RBMY1, CDY1, and VCY2 genes are examined in testicular biopsy specimens from 42 Korean azoospermic patients. No expression of RBMY1, CDY1, and VCY2 genes appeared as 34%, 66%, and 27% of the male infertility, respectively. Patients who had no expression of RBMYl and VCY2 genes also showed negative expression of the CDY1 gene in their testis tissues. All Sertoli cell-only syndrome patients showed no expression of the CDY1 gene. Taken together, the CDY1 gene expression seems to be necessary factor to complete spermatogenesis in Korean population.