Transcriptional regulation of mammalian LTR-retrotransposon element on the human Dorfin geneJa-Rang Lee1, Jae-Won Huh1, Dae-Soo Kim2, Kung Ahn1, Hong-Seok Ha1, Yun-Ji Kim1, Won-Ho Lee1, and Heui-Soo Kim1,2,*

1Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea2 PBBRC, Interdisciplinary Research Program of Bioinformatics, Pusan National University, Busan 609-735, Korea

AbstractDorfin containing RING-finger and IBR motifs is an E3 ubiquitin ligase that is localized in Lewy bodies, a characteristic neuronal inclusion in Parkinson’s disease brains. The Dorfin gene located on human chromosome 8q22.2 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 MaLR (mammalian LTR-retrotransposon) element using bioinformatic tools. The MaLR-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 MaLR element on Dorfin gene indicated good activity in human colon carcinoma cells (HCT-116). These findings suggest that the MaLR element acquired the role of transcriptional regulation of Dorfin gene during primate evolution..

Introduction- cloned from the anterior horn tissues

category research

genome

transcriptome

proteome

Not searched in genome level

cDNA sequence decisionExpression pattern identification by nothern blotTranscripts expression research in ALS

Identification of E3 activityLocation decision of Dorfin in cellPhylogeny research in RBRfamilyHomology research related with parkinResearch about activity mechanism (VCP releated)

Parkinson’s disease Amyotrophic lateral sclerosis

NM_015435.3 

Other region16%

Gene-related Sequence

36%

LINE20%

SINE13%

Coding sequence3%

Pseudogene1%

HERV element8%DNA element

3%

Materials & MethodsLuciferase assay

Gene cloning

RT-PCR

Primer Design

Twenty Different Human tissue cDNAs

QuantitativeAnalysis

Real-time PCR

LuciferaseLuciferaseassayassay

Modified PGL2-vector, HCT116, Cos7,

Lipofectamine, Dual luciferase Assay

Gene Gene cloningcloning

Qiaquick Gel-extraction, Promega T-easy Vector,

EcoR-I(DH5α)

RT-PCRRT-PCR35cycle

94 : 40sec 56 : 30sec 72 : 40sec℃ ℃ ℃ .

Real-timeReal-timePCRPCR

40cycle (Sybr green)

94 : 10sec 56 : 15sec 72 : 15sec℃ ℃ ℃

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]

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.

Results & Discussion

Human

Dog

Pig

Cattle

Mouse

Rat

Chicken

Zebrafish

100

100

100

42

77

0.05

100

77

42

100

100

100

Human

Dog

Pig

Cattle

Mouse

Rat

Zebrafish

Chicken

8

Dorfin(RNF19)

S18q22.2

MaLR

1 2 3 4 5 6 7 8 9 101a 1b

AS2S2

AluJ

AS1

RING-finger/IBR

ORF

S3 AS3

Dorfin gene structure

Mar

ker

Adr

enal

gla

nd

Adu

lt br

ain

Fet

al b

rain

Fet

al li

ver

Hea

rt

Kid

ney

Liv

er

Lun

g

Bon

e m

arro

wC

ereb

ellu

m

Skel

etal

mus

cle

Mar

ker

Pla

cent

a

Spin

al c

ord

Tes

tis

Thy

mus

Thy

roid

Tra

chea

Ute

rus

Pro

stat

e

Dorfin

Gapdh

494 bp

195 bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Bre

ast

(N)

Bre

ast

(C)

Mar

ker

AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC

R LE GN G DV IG A

R LE GN G DV IG A IS

GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC

CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG

CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG

TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT

TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT

TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA

GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA

CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG

CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA

ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC

AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC

AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA

GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC

CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG

CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG

TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT

TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT

TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA

GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA

CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG

CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA

ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC

Primer S2

Primer AS2

M Q E Q E I G F S KI Y

G

N E

CL NV T PD SV I TL IS L D

M LS RH Q GM DS R LD SQ S

A SS SV L SP KV K PA KK R R

R SI GI S FL KR K ND RK K S

IS SE I

M LS RH Q GM DS R LD SQ S

A SS SV L SP KV K PA KK R R

R SI GI S FL KR K ND RK K S

SE I

AluJ element

AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA

556 bp430 bp315 bp

(A) (B)

(C)3 111 2

MaLR AluJ

AluJMaLR

2 101

AluJMaLR

2 111 3

126 bp 115 bp

1)

2)

3)

123

0

0.01

0.02

0.03

0.04

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

MaLR-derived promoter transcript (556 bp)

MaLR-derived promoter transcript (430 bp)

Rel

ativ

e E

xpre

ssio

n

Bone marrow

Cerebellum

Kidney

Lung

Testis

Reverse

Forward

Reverse

Forward

Control

Relative Luciferase Activity

(Fold of pGL-2 control)

0 2 4 6 8 10 12 14 16 18 20 22

Cos7

HCT116

CCCCACAAAA GATATGTTCA TGTCCTAATC CCCAGAATCT GCAAATGTTA TTTGGAAAAA

GGGGTTTTGC AGATGTAATT AAGTTAAGAA TCTTGAGATA AGATCATCCT GGATTATCCA

GGTAGCCTCA AAATCAAGTG ACAAGTGTCT TTGTAAGGGA CAAGTAGACC CATTACAGAG

AAGACGACGC GCAGAAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG

TAACCGCAAG CCGAGGAATG CTTATAGTCA CCAGAAGCTG GAAGAGTCAA AGGACACATT

CTCCCCTCAA GCCCCAGTGG GAGCACGGCC CAGCTGGATT TTGGACTTCT GGCCTCCAGA

ACTGTAAGAG AAATGTCCAT TGTCTTAAGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG

CCCCAGGAAA CTACTA .

GATA-1

GATA-2

Nkx2-5 Nkx2-5

Whn ELF-1

+1 TRANSCRIPTION START SITE

In silico analysis of transcription binding sites of MaLR element

References1. 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.