Functional HaeIII DNAs are bona fide transcriptional regulatory elements in F9 cells. (A) Proximal HaeIII fragments represent activating components of endogenous promoters in F9 cells. (Left panel) Luciferase expression exhibited by each of the plasmid constructs (right) relative to the pGL3 vector is indicated on the Y-axis. (Right panel) Plasmid constructs. The gray rectangles indicate the locations of the NFR-derived HaeIII fragments 27b and 2-52 relative to the associated genes’ TSS. These regions are deleted in constructs D27b and D2-53. (B) Luciferase assays to assess the ability of the HaeIII DNAs to act as enhancers. Seventeen distal elements identified in the GFP screen were cloned 2 kb downstream of the TK promoter in the pTK-luciferase reporter plasmid and transfected into F9 cells. Control plasmid 2-320 (highlighted by the gray box) contains a proximal element insert; FgfENH contains the Fgf4 enhancer to provide a positive control. Values were averaged and normalized to the luciferase activity of pTK-luciferase (assigned the value 1) and are expressed as fold induction relative to pTK-luciferase. Results for enhancers exhibiting moderate (left panel) or high (right panel) activity are shown. (C) Active HaeIII DNAs localize to genomic regions enriched for H3K4me3 in ES cells. The genomic coordinates (mm8 assembly) for each of the HaeIII DNAs were submitted to http://www.broad.mit.edu/seq_platform/chip/ to visualize their position relative to nucleosomes containing modifications of Histone H3. The HaeIII element analyzed is indicated on the lower left of each panel. “Random” indicates a representative HaeIII DNA sequence derived from the in silico HaeIII digestion of genomic mouse DNA. 2-320 is representative of the result observed for analysis of proximal elements and is highlighted in yellow. The remaining HaeIII elements are distal elements (Supplemental Table 2). Each panel shows an ∼100-kb segment containing the HaeIII element, and genomic locations of H3K4me3 are indicated in green. The red line indicates the position of the each of the HaeIII elements. The width of this line is not drawn to scale (the average length of these elements is 149 bp). The complete relationship between H3K4me3 and all of the elements is shown in Supplemental Figure 3.
