Figure 1.

The Sfpi1fragment CE5-CE3 is a myeloid restricted enhancer. (a) A schematic showing the UCSC mammalian conservation track with genome alignment of the ~18 kb upstream region of the Sfpi1 gene is shown. Regions used in reporter constructs are depicted. Reporter constructs were designed as described previously (14). (b) Transient transfection assays showing Sfpi1 reporter activity in NFS-25 pre-B-cells and RAW264.7 macrophages. Data represent the average fold difference relative to L1. NFS-25 data are from five independent experiments. RAW264.7 data shown are from a single representative experiment performed in triplicate. Error bars represent standard deviations. Cells were transfected using FuGENE at a 3:1 DNA:FuGENE ratio. NFS-25 cells were grown in RPMI media with 10% fetal bovine serum, penicillin/streptomycin/glutamine, non essential amino acids, sodium pyruvate, and 50 μM 2-mercaptoethanol. RAW264.7 cells were grown in DMEM media with 10% fetal bovine serum and penicillin/streptomycin/glutamine. Transfected cells were harvested 30-48 hours post transfection. Cells were cotransfected with pRL-CMV and lysates were analyzed using Promega’s Dual Luciferase system. (c) The CE5-CE3 fragment is a myeloid specific enhancer when integrated into chromatin. Cell lines were stably transfected with the L9-6 and L9-3 constructs depicted in Fig 1A. Bars show the geometric mean of six independent mixed pools (dots) of stably transfected cells 30 days post transfection. Data are reported as relative light units (RLU). P-value is from student’s T-test of log₁₀ transformed data. FuGENE:DNA complexes were formed then the same complexes were aliquot to 6 well plates containing either NFS-25 or RAW264.7 cells. For stable transfections, Sfpi1 reporters were linearized with Not I prior to transfection. The renilla luciferase was cloned into Invitrogen’s pTracer EF/Bsd A and the construct was linearized with Fsp I for cotransfection with Sfpi1 reporters. Cells were selected with 5-15 μg/ml of Blasticidin for their duration in culture, beginning one day post transfection. (d) Multigenome alignments and transcription factor target site prediction analysis of the CE5 and CE7/6 regions are shown. TRANSFAC analysis was performed through the Biobase TRANSFAC suite’s MATCH tool (https://portal.biobase-international.com/cgi-bin/portal/login.cgi). Predicted hematopoietic transcription factors with matrix similarity matches above 0.9 are shown (black). Some matches below 0.9 are also shown (gray). CBF = Core Binding Factor sites for the Runx family. “Ets” labeled sites are general Ets family sites that potentially bind to multiple Ets family factors. Asterisks mark conserved sites present in only 4/6 aligned sequences. All other sites are present in all six sequences. The boxed sequence shows a region of CE5 with a PU.1 target site adjacent to a specifically predicted Ikaros target site. The red bar represents the DNA probe CE5-P2 designed for gel shift assays (Fig. 3).