Alternative splicing and promoter of the GSDML gene implicated in the HERV-H LTR element Kung-Ahn, Jae-Won Huh, Dae-Soo Kim, Hong-Seok Ha, Yun-ji Kim, Ja-rang Lee, Heui-Soo Kim aDivision of Biological Sciences, College of Natural Sciences, Pusan National University, Busan , Korea b PBBRC, Interdisciplinary Research Program of Bioinformatics, Pusan National University, Busan , Korea endogenous retroviruses (HERVs) are the remnants of ancient exogenous retrovirus infection. Long terminal repeats (LTRs) of HERVs have the potential to control the adjacent gene. A new transcript variant of the GSDML (gasdermin-like protein)gene was formed by the integration of the HERV-H LTR element during hominoid evolution. Here, we use bioinformatics tools to identify nine transcript variants of the GSDML gene containing the HERV-H LTR element. The variants appeared in various human tissues in different ways. Binding sites for the potential transcription factor of the LTR promoter region were identified by in silico analysis. In the transient transfection assay, deletion of the 5’ flanking region of LTR sequences led to a threefold increase compared to thefull-length clone, while deletion of the U5 region showed a critical decrease of transcription activity. Taken together, we suggest that the HERV-H LTR element of GSDML could be a goodsource for transcriptional diversification by providing a novel promoter.. In silico analysis for alternative splicing of the GSDML gene Modification of the pGL2-basic vector for the transient transfection assay Cloning of deletion mutants Cell culture Transfection, and Luciferase Assay, Experimental Procedure REFERENCES 1.Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, W., Miller, W., Lipman, D.J., Gapped Blast and PSI- BLAST: a new generation of protein database search program. Nucleic Acids Res. 25, Baban, S., Freeman, J.D., Mager, D.L., Transcripts from novel human KRAB zinc finger gene contain spliced Alu and endogenous retroviral segments, Genomics 33, de Parseval, N., Alkabbani, H., Heidmann, T., The long terminal repeats of the HERV-H human endogenous retrovirus contain binding sites for transcriptional regulation by the Myb protein. J. Gen. Virol. 80, Dunn, C.A., Mager, D. L., Transcription of the human and rodent SPAM1 / PH-20 genes initiates within an ancient endogenous retrovirus, BMC Genomics 6, Dunn, C.A., Romanish, M.T., Guiterrez, L.E., van de Lagemaat, L.N., Mager, D.L., Transcription of two human genes from a bidirectional endogenous retrovirus promoter. Gene 366, ABSTRACT MATERIALS & METHODS INTRODUCTION RESULTS & DISCUSSION A representation of various GSDML gene transcript variants.. Boxed arrows indicate the genomic locus of the HERV-H LTR element. Open asterisks indicate the full-length, mRNA-derived transcript variants. Dotted boxes represent expected exon positions.. Identification of nine transcript variants of the GSDML gene Expression analysis of the GSDML gene Dissection of the HERV-H LTR promoter region and deletion mutants A representation of the reporter gene used for transient transfection from the deletion mutants of the GSDML gene promoter region. Small arrows indicate the tandem repeat sequences in the U3 region. The U5, R, and U3 regions are indicated by the dotted line... Combined expression analysis of the LTR- derived transcript and the original transcript using an experimental approach and an in silico analysis.. Open boxes indicate no expression; creased line boxes indicate expression in tested human normal tissues from our previous experimental results. Closed boxes indicate the expression of tested transcript from the in silico analysis.... The GSDML gene is located in the GSDML-GSDM locus on human chromosome 17q21 linked to the oncogenomic recombination hotspot around the PPP1R1B-ERBB2-GRB7 amplicon. The HERV-H LTR promoter of GSDML showed bidirectional transcription activity through the transient transfection assay in human cancer cell-lines HCT-116 and HeLa, and kidney cell line Cos7 of the African green monkey. Interestingly, the LTR promoter showed stronger transcription activity with the antisense orientation than it did with the sense orientation In the present study, nine transcription variants, the expression, and the evolution of the GSDML gene were analyzed. The critical region for transcription regulation of the HERV- H LTR element in GSDML was explored using six different deletion mutants and the dual-luciferase reporter assay. The LTR-derived transcript is expressed in various tissues (brain, prostate, testis, kidney, placenta, skeletal muscle, spleen, thymus, and uterus), whereas expression of the original transcript is restricted to normal stomach tissues only HERVs have been regarded as remnants of ancient exogenous retrovirus infection and have been implicated in multiple sclerosis, schizophrenia, and autoimmune rheumatism. Since the surprising finding of the connection of the syncytin molecule (HERV-W env element) with placental morphogenesis, many researchers have focused on the biological role of various HERV elements.In various primate lineages, tissues, and cell lines, HERV elements have been identified and analyzed by molecular and bioinformatics. As the integration of the majority of HERV elements during primate evolution is recent, their existence or presence could have an effect on speciation and diversification. Most HERV families were integrated into our common ancestor genome after the divergence of New World monkeys and Old World monkeys, and subjected to amplification several times in the primate genome. Their structure was rendered defective by multiple stop codons, deletions and insertions. Nevertheless, some structural genes of HERV elements are expressed actively in human placenta and various cancer cell-lines. One of the most abundant HERV families, HERV-H, has appeared in the New World monkey lineage but it was expanded mainly in the Old World monkey. S Sequence and structure analysis of the GSDML gene promoter region