Transcriptional regulation of mammalian LTRretrotransposon element on the

Transcriptional regulation of mammalian LTR-retrotransposon element on the human Dorfin gene Ja-Rang Lee 1, Jae-Won Huh 1, Dae-Soo Kim 2, Kung Ahn 1, Hong-Seok Ha 1, Yun-Ji Kim 1, Won-Ho Lee 1, and Heui-Soo Kim 1, 2, * of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609 -735, Korea PBBRC, Interdisciplinary Research Program of Bioinformatics, Pusan National University, Busan 609 -735, Korea Abstract Results & Discussion Dorfin containing RING-finger and IBR motifs is an E 3 ubiquitin ligase that is localized in Lewy bodies, a characteristic neuronal inclusion in Parkinson’s disease brains. The Dorfin gene located on human chromosome 8 q 22. 2 has showed 4. 4 kb transcript and expressed ubiquitously in various tissues including brain. Here we found its alternatively spliced transcript variants which derived from Ma. LR (mammalian LTR-retrotransposon) element using bioinformatic tools. The Ma. LR-derived promoter transcripts are detected as two different types in all tissues examined, while breast tissue only showed three variant types. Reporter gene assay of the promoter activity of Ma. LR element on Dorfin gene indicated good activity in human colon carcinoma cells (HCT-116). These findings suggest that the Ma. LR element acquired the role of transcriptional regulation of Dorfin gene during primate evolution. . D 100 e 8 Human 100 en g n i orf re tu c u r t s Dog 77 Pig 42 Cattle Mouse 100 RING-finger/IBR Rat 100 1 a 1 Chicken Zebrafish 8 q 22. 2 Dorfin (RNF 19) Ma. LR S 3 Amyotrophic lateral sclerosis NM_0154 35. 3 LINE 5 6 7 8 9 ORF AS 2 AS 3 us Uter ea Trac h roid Thy m Test is al co rd Ske us scle l mu leta ate Pros t nta Plac e Mar ker g Live Lun r ey Kidn Hea rt Feta l liv er l bra in ain lt br Adu Dorfin 494 bp 1 16% m Identification of E 3 activity Location decision of Dorfin in cell Phylogeny research in RBRfamily Homology research related with parkin Research about activity mechanism (VCP releated) Cere bellu proteome w c. DNA sequence decision Expression pattern identification by nothern blot Transcripts expression research in ALS em arro transcriptome Bon Not searched in genome level land genome Other region 13% 4 research Mar ker Adr ena lg category 3 Alu. J S 2 - cloned from the anterior horn tissues SINE 2 AS 1 Introduction Parkinson’s disease 1 b S 1 Spin 2 1 Division 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Gapdh 195 bp 20% HERV element Gene-related Sequence DNA element Pseudogene 8% 36% Marker Coding sequence Primer S 2 (B) 3% A G A A G C T G G A A G A G T C A A A GG A C A C A T T C T C C C C T C A A G CC C C A G T G G G A Breast (C) 1% (A) Breast (N) 3% G C A C G G C C C A G C T G G A T T G G A C T T C T G G C C T C C A G A A C T A G A C A G G G C A C G G C C C A G C T G G A T T G G A C T T C T G G C C T C C A G A A C TA G A C A G G G C C T C A C T G T G T C A C C C A G G G T G G A A T A C A G T G T G A T C A T A G C T C A C T G Alu. J element C A G C C T G G A A T T C C T G G G C T C A A G C A A C C C T G C C A C C T C A G C C T T C C A A G T T T C A A G A A T T C T G A A G G A A C C A A G T A G G A T T C T T A C A T G A C T T C A A G A A T T C T G A A G G A A C C A AG T A G G A T T C T T A C A T G A C T M RT-PCR Luciferase assay Modified PGL 2 -vector, HCT 116, Cos 7, Lipofectamine, Dual luciferase Assay 1 Ma. LR 2 Ma. LR 11 Alu. J 2 10 I F K S Y N E L Ma. LR V C T N P D V I S L S T I D L M S L H R Q M G S D R D L Q S S Testis Cerebellum Kidney Bone marrow C A T G A G T T T A C A T C G G C A A A T G G G T T C A G A T C G A G A T C T T C A G T C C T C T G S L S V S Q L P S S V K K A P Q K S R R C T T C A T C T G T G A G C T T G C C T T C A G T C A A A A A G G C A C C C A A A A G A 2 11 3 S R S I V S S I L G P S S L V F K R K K A K P D K N K K R R R K S A T T T C A A T A G G C T C C C T G T T T C G G A A A A A A G A T A A C A A A C G T A A A T C R Alu. J I S I G S L F R K K D N K R K S R E L N G G V D G I A S I E S I A A G G G A G C TA A A T G G C G G G G T G G A T GG A A T T G C A A G T A T T G A A A G T A T A C Qiaquick Gel-extraction, Promega T-easy Vector, Eco. R-I(DH 5α) G A 3) G I E Q G G G C T G T G T A A A C T G A C C C T G T C A A T T C T A A C A A G C A T T A G G G C T G T G T A A A C T G A C C C T G T C A A T T C T A A C A A GC A T T A G A A Alu. J 1 Gene cloning 3 1 2) E Q T A A T G T G A A T G C A A G A A A T A G G T T T T A T C T C T A A A T A T A A T G A A (C) 1) Luciferase assay T A G C T A G G A C T A C A G A A C A T C C A T G A T A G C A G T C T G T A A A T C G A A C T T A G C T A G G A C T A C A GA A C A T C C A T G A T A G C A G T C T G T A A A T C G A A C T Relative Expression Materials & Methods 1 2 3 556 bp 430 bp 315 bp Lung 126 bp 115 bp Ma. LR-derived promoter transcript (556 bp) Primer AS 2 Ma. LR-derived promoter transcript (430 bp) Real-time PCR Gene cloning Primer Design RT-PCR 35 cycle 94 ℃ : 40 sec 56 ℃ : 30 sec 72 ℃ : 40 sec. In silico analysis of transcription binding sites of Ma. LR element GATA-1 CCCCACAAAA GATATGTTCA TGTCCTAATC CCCAGAATCT GCAAATGTTA TTTGGAAAAA GATA-2 Reverse Twenty Different Human tissue c. DNAs Quantitative Analysis Real-time PCR 40 cycle (Sybr green) GGGGTTTTGC AGATGTAATT AAGTTAAGAA TCTTGAGATA AGATCATCCT GGATTATCCA Cos 7 Nkx 2 -5 Forward 94 ℃ : 10 sec 56 ℃ : 15 sec 72 ℃ : 15 sec GGTAGCCTCA AAATCAAGTG ACAAGTGTCT TTGTAAGGGA CAAGTAGACC CATTACAGAG Whn HCT 116 References 1. Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006. Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells. Arch. Virol. [Epub ahead of print] 2. Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003. Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and amyotrophic lateral sclerosis. Am J Pathol. 163(2): 609 -619. Nkx 2 -5 Reverse ELF-1 AAGACGACGC GCAGAAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG +1 TRANSCRIPTION START SITE TAACCGCAAG CCGAGGAATG CTTATAGTCA CCAGAAGCTG GAAGAGTCAA AGGACACATT Forward CTCCCCTCAA GCCCCAGTGG GAGCACGGCC CAGCTGGATT TTGGACTTCT GGCCTCCAGA Control Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006. Transcriptional control of the HERV-H LTR element of the GSDML gene in ACTGTAAGAG AAATGTCCAT TGTCTTAAGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG 0 2 4 6 8 10 12 14 16 18 human tissues and cancer cells. Arch. Virol. [Epub ahead of print]20 22 Relative Luciferase Activity (Fold of p. GL-2 control) C C C A G G A A A C T A. Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003. Dorfin Clocalizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and amyotrophic lateral sclerosis. Am J Pathol. 163(2): 609 -619. 10