title: Sleeping beauty transposase has an affinity for heterochromatin conformation.
abstract: The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4',6'-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.
authors.affiliation: Department of Social and Environmental Medicine H3, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. takeda@mr-envi.med.osaka-u.ac.jp
authors.forename: Ryuji
authors.initials: R
authors.lastname: Ikeda
authors.forename: Chikara
authors.initials: C
authors.lastname: Kokubu
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yusa
authors.forename: Vincent W
authors.initials: VW
authors.lastname: Keng
authors.forename: Kyoji
authors.initials: K
authors.lastname: Horie
authors.forename: Junji
authors.initials: J
authors.lastname: Takeda
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D004251:DNA Transposable Elements / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology; D005456:Fluorescent Dyes; D020868:Gene Silencing; D006570:Heterochromatin / Q000737:chemistry / Q000378:metabolism*; D007211:Indoles; D051379:Mice; D016254:Mutagenesis, Insertional; D041681:NIH 3T3 Cells; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection; D019895:Transposases / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D005456:Fluorescent Dyes; D006570:Heterochromatin; D007211:Indoles; C007293:DAPI; D019895:Transposases