Alternative splicing and promoter of the Alternative splicing and promoter of the GSDML GSDML gene implicated in gene implicated in the HERV-H LTR element the HERV-H LTR element Kung-Ahn, Jae-Won Huh , Dae-Soo Kim, Hong-Seok Ha, Yun-ji Kim, Ja-rang Lee, Heu i-Soo Kim aDivision of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea b PBBRC, Interdisciplinary Research Pr ogram of Bioinformatics, Pusan National University, Busan 609-735, Korea endogenous retroviruses (HERVs) are the remnants of ancie nt exogenous retrovirus infection. Long terminal repeats (LTRs) of HERVs have the potential to control the adjacen t gene. A new transcript variant of the GSDML (gasdermin- like protein)gene was formed by the integration of the HE RV-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 w ays. Binding sites for the potential transcription factor of the LTR promoter region were identified by in silico a nalysis. In the transient transfection assay, deletion of the 5’ flanking region of LTR sequences led to a threefol d increase compared to thefull-length clone, while deleti on of the U5 region showed a critical decrease of transcr iption activity. Taken together, we suggest that the HERV -H LTR element of GSDML could be a goodsource for transcr iptional diversification by providing a novel promoter. . In silico analys is for alter native splic ing of the G SDML gene Modificatio n of the pG L2-basic ve ctor for th e transient transfectio n assay Cloning of deletion mutants Cell culture Transfection, and Lucife rase Assay, Experimental Procedure Http://www.primate.or.kr REFERENCES 1. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, W., Miller, W., Lipma n, D.J., 1997. Gapped Blast and PSI- BLAST: a new generation of protein database search program. Nucleic Acids Res. 25, 3389-3402. 2. Baban, S., Freeman, J.D., Mager, D.L., 1996. Transcripts from novel human KRAB z inc finger gene contain spliced Alu and endogenous retroviral segments, Genomics 33, 4 63-472. 3. de Parseval, N., Alkabbani, H., Heidmann, T., 1999. The long terminal repeats of the HERV-H human endogenous retrovirus contain binding sites for transcriptional regulatio n by the Myb protein. J. Gen. Virol. 80, 841-845. 4. Dunn, C.A., Mager, D. L., 2005. Transcription of the human and rodent SPAM1 / PH -20 genes initiates within an ancient endogenous retrovirus, BMC Genomics 6, 47. 5. Dunn, C.A., Romanish, M.T., Guiterrez, L.E., van de Lagemaat, L.N., Mager, D.L., 2006. Transcription of two human genes from a bidirectional endogenous retrovirus promoter. Gene 366, 335-342. 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 v ariants. Dotted boxes represent e xpected 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 g ene used for transient transfectio n from the deletion mutants of the GSDML gene promoter region. Small arrows indicate the tandem r epeat sequences in the U3 region. The U5, R, and U3 regions are indi cated by the dotted line. .. Combined expression analysis of the LTR-derived transcript and the orig inal transcript using an experiment al approach and an in silico analysis. . Open boxes indicate no expression; creased line boxes indicate express ion in tested human normal tissues from our previous experimental resu lts. Closed boxes indicate the expr ession of tested transcript from th e 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 PPP1R 1B-ERBB2-GRB7 amplicon. The HERV-H LTR promoter of GSDML showed bidirec tional transcription activity through the transi ent transfection assay in human cancer cell-line s 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 us ing six different deletion mutants and the dual- luciferase reporter assay . The LTR-derived transcript is expressed in var ious tissues (brain, prostate, testis, kidney, placenta, skeletal muscle, spleen, thymus, and uterus), whereas expression of the original tr anscript is restricted to normal stomach tissu es only HERVs have been regarded as remnants o f ancient exogenous retrovirus infection and h ave been implicated in multiple sclerosis, sch izophrenia, and autoimmune rheumatism . Since the surprising finding of the connection of th e syncytin molecule (HERV-W env element) with p lacental 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 identi fied and analyzed by molecular and bioinformat ics. As the integration of the majority of HER V elements during primate evolution is recent, their existence or presence could have an effe ct on speciation and diversification. Most HER V families were integrated into our common anc estor genome after the divergence of New World monkeys and Old World monkeys, and subjected t o amplification several times in the primate g enome. Their structure was rendered defective by multiple stop codons, deletions and inserti ons. Nevertheless, some structural genes of HE RV elements are expressed actively in human pl acenta and various cancer cell-lines. One of the most abundant HERV fam ilies, HERV-H, has appeared in the New World m onkey lineage but it was expanded mainly in th e Old World monkey . S Sequence and structure analys is of the GSDML gene promoter region