Identification And Characterization Of Three Novel Human Endogenous Retrovirus-related Genes

Human endogenous retroviruses(HERVs) are genomic DNA sequences that are homologous to retroviruses in humans.They are remnants of ancient retroviral infections of the human genome.Most of the HERV open reading frames(ORFs) have been degraded by deletion or mutation during evolution as part of the human genome. However,there are reports indicating that abnormal expression of HERVs is associated with diseases including cancer.In a previous study using microarray to identify related genes in disease tissues,a 586-bp HERV-related sequence was found to be specifically up-regulated in colon tumor samples.This finding led us to study on the most homologous gene psiTPTE22,which has been defined as a pseudogene and encodes some HERV-related transcripts.In this study,psiTPTE22 was found to be not a colon cancer-related gene.However,two novel genes that differentially express in tumor and normal colon tissues were identified,including HERV-HX and HERV-H4p15.2.Further study on the so-called pseudogene identified a new functional gene(psiTPTE22-HERV) that is negatively associated with kidney cancer.All three new genes consist of HERV sequences.Identification and characterization of these three HERV-related genes are contained in this dissertation.Using RT-PCR to measure psiTPTE22 expression levels,no significant difference was found between colon tumor and normal tissues,indicating it is not a colon cancer-related gene detected by the 586-bp microarray sequence.However,using RT-PCR primers targeting the HERV part of psiTPTE22,some non-target gene products were amplified specifically in colon tumor samples.Further study on these colon tumor-related PCR products identified a novel gene HERV-HX,which is an H family HERV(HERV-H) located on chromosome X.The full-length transcript of HERV-HX was obtained by RACE assays and deposited in GenBank with accession number EF194101.In situ hybridization and quantitative RT-PCR confirmed its expression was specifically up-regulated in colon tumor samples.Sequence analysis revealed that large fragments are missing in the HERV-HX provirus,and the sequence deleted in the env region corresponds to the env ORF.Using RT-PCR to analyze the transcription of the env-deleted HERV-HX and other env-intact HERV-H elements,it was demonstrated that transcription of HERV-H elements in colon cancer is not associated with the env gene.Promoter activity assays indicated that a 17-bp sequence within the U3 region of the 5' long terminal repeat is important to the expression of HERV-HX.Knock-down of HERV-HX by RNA interference reduced proliferation and increased apoptosis in colon cancer HT29 cells.These findings suggest that HERV-HX is a novel colon cancer-related gene,and it may play an important role in colon carcinogenesis.The psiTPTE22 gene,located on chromosome 22ql 1.2,has been designated as a TPTE pseudogene.We found the 5' part of psiTPTE22 had no sequence similarity to TPTE and contained a HERV-H element.Because of the HERV element,the 5' part of psiTPTE22 expressed as an independent human specific gene,and we defined it as psiTPTE22-HERV.Three transcript variants of psiTPTE22-HERV were identified by RT-PCR and RACE assays and deposited in GenBank with accession numbers EU541207,EU541208,and EU541209.RT-PCR and quantitative RT-PCR results showed psiTPTE22-HERV was expressed at high levels in the normal samples and down-regulated in paired tumor samples of kidney,liver,stomach,and lung.Bisulfite sequencing PCR results indicated that it was silenced in cancer cells by DNA methylation.The expression of psiTPTE22-HERV was recovered in cancer cells by treatment with DNA methylation and histone deacetylase inhibitors.A protein product was found to be translated from psiTPTE22-HERV and mainly localized in the cytoplasm of cells.Knock-down of psiTPTE22-HERV inhibited proliferation of human embryonic kidney 293T cells,accompanied by G1 phase arrest and increase in apoptosis,suggesting psiTPTE22-HERV plays critical roles in cell growth and its silencing might not be a primary cause of cancer.Treating cells with 5-fluorouracil after knocking down or over-expressing psiTPTE22-HERV,it was demonstrated that psiTPTE22-HERV levels correlated with the sensitivity of 293T cells to 5-fluorouracil. psiTPTE22-HERV,expression of which is negatively associated with cancers,is a novel human specific gene with complex functions.Using RT-PCR and 3' RACE to study psiTPTE22-HERV,two new sequences were identified incidentally and deposited in GenBank with accession numbers EF535612 and EF535613.Sequence analysis of these two sequences revealed that they were from a novel HERV-H-related gene.Thus 5′RACE was further performed to identify the 5′terminal sequence.The full-length RNA sequence has been deposited in GenBank with accession number EU669866.Located on chromosome 4p15.2,DNA sequence of this gene consists of a 5.0-kb structurally incomplete HERV-H element and an Alu element immediately upstream.The 1.3-kb full-length RNA consisting of four exons contains no putative ORFs,and may be an mRNA-like non-coding RNA.RT-PCR and quantitative RT-PCR results indicated that expression of this HERV-related RNA was down-regulated in tumor samples of colon,stomach and kidney compared to their adjacent normal samples.Its expression was recovered by treatment with DNA methylation and histone deacetylase inhibitors in colon cancer cells,suggesting it might be regulated epigenetically.Each of the three novel genes identified in this study has its own distinguishing characteristics.They are important for the study of HERV-related genes and our understanding of the human genome.Elucidation of their functions will contribute to a better understanding of the mechanisms of cancer development.