title: LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of beta -globin intron-2.
abstract: Human beta-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5'HS3 element requires the presence of the AT-rich region (ATR) in beta-globin intron-2. Here, we examine the dependence of 5'HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5'HS3 beta/gamma-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P <.05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P <.05). We conclude that the ability of 5'HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5'HS3 beta/gamma-globin transgene expression and should be retained in LCRbeta-globin expression cassettes designed for gene therapy.
authors.affiliation: Developmental Biology Program, Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Rikki R
authors.initials: RR
authors.lastname: Bharadwaj
authors.forename: Cecelia D
authors.initials: CD
authors.lastname: Trainor
authors.forename: Peter
authors.initials: P
authors.lastname: Pasceri
authors.forename: James
authors.initials: J
authors.lastname: Ellis
mesh_terms: D020856:AT Rich Sequence / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D051863:Host Cell Factor C1; D007438:Introns; D019901:Locus Control Region / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D014157:Transcription Factors; D005914:Globins; D004247:DNA