A MIR repeat within the +18 region boosts the activity of the Tal1 +19 enhancer. (A) Mouse/human nucleotide sequence of the +18 region aligned to the MIR consensus sequence, in which the A and B boxes of the MIR tRNA region are underlined. The nucleotide sequence contained within the boxed region denotes the 200 bp of the +18 region that was deleted in the SV/Luc/+18,19ΔMIR construct, and the conserved sequence that was mutated to a BglII restriction site for SV/Luc/+18,19M1 is marked. (B) Mutation analysis of the +18 region. Shown on the left are the luciferase reporter constructs depicting the deletion in the +18 region and the mutation in the first block of sequence conservation highlighted in A. The mutation constructs were analyzed in 416B stable transfection assay and the luciferase values presented as fold increase over the SV40 luciferase control, which was assigned a value of 1. Data are presented as the mean luciferase activity of three biological replicates performed in quadruplicate plus or minus the standard error of the mean. (C) In vivo DMS footprinting of the +18 MIR repeat region (antisense strand). Protections (◦) were observed over the M1 region of the MIR repeat in two independent 416B samples compared to the naked genomic control. (D) Shown on the left are the luciferase reporter with constructs, either the heterologous SV40 enhancer or the tissue-specific Lyl1 promoter proximal enhancer, inserted downstream from the SV40 luciferase reporter cassette with and without the +18 region. The luciferase activities are shown as fold increase over SV40 luciferase with either the SV40 enhancer or the Lyl1 enhancer, each of which was assigned a value of 1.
