FIG. 1.

The Sfpi1 upstream region contains novel cis-regulatory elements. (A) Sfpi1 multigenome alignment from exon 1 to ∼15 kb upstream. Schematics of regions used in reporters are shown. (B and C) The CE9 to CE8 (URE) region is an enhancer in the RAW 264.7 myeloid cell line and in the immature DN3-like Adh.2C2 pro-T-cell line. The Renilla luciferase-expressing control vector pRL-CMV, was used as an internal standard. The empty pGL3-basic vector was used as a control (LB). The average relative light units (RLU) of triplicates from a representative experiment are shown with standard deviations. (D) The CE9 to CE8 region does not possess enhancer activity in a mature T-cell line, EL4. Data shown are the averages of three independent experiments performed in duplicate with standard deviations. (E) Novel DNase I HS sites were identified. Probe 2 Southern blotting results for SphI-digested DNA from nuclei of indicated cell lines, with or without DNase I, are shown. For sites at and downstream of promoter, see Fig. 2. Bands are defined by sites of DNase I sensitivity. The right panel, more extensively digested, shows a T-cell-specific doublet of HS sites at ∼2 kb from SphI site (box). (F) A schematic summarizing HS mapping with various probes. S, SphI; H, HindIII; E, EcoRV. (G) The L5-3 reporter showed lineage-specific activity. RLUs were normalized to L1. The average RLU of triplicates from a representative FuGENE transfection experiment are shown, with standard deviations.