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. 2017 Nov 15;19(1):118–134. doi: 10.15252/embr.201744201

Figure 5. T regulates Lmo2 expression through binding to a distal enhancer.

Figure 5

  1. T binding and H3K27ac/me3 tracks from T WT and T Y88A mutant mesodermal cells differentiated in vitro at the Lmo2 locus. The black arrow indicates the T binding region that was removed using CRISPR/Cas9 nickase technology. Red lines indicate primers used to genotype the CRISPR/Cas9‐mediated deletion. The yellow box indicates altered H3K27ac at the TSS. The purple‐shaded box indicates the region amplified in the ChIP‐qPCR analysis.
  2. ChIP‐qPCR analysis of H3K27ac at the −70 enhancer of Lmo2. IgG ChIP was used as a negative control. Error bars depict SD of n = 3–4 biological replicates. *P = 0.009 using a one‐sample t‐test.
  3. Genotyping PCR from genomic DNA of Lmo2‐70 ΔTBS/ΔTBS mESC clones, using primers depicted in (A), revealed homozygous mutant deletion clones.
  4. Motif analysis of the 400‐bp deleted region within the −70 enhancer of Lmo2. Colored boxes represent motifs pictured below, as predicted by ConSite 60.
  5. Table illustrating the lack of Sox17 and Runx1 expression in RNA‐Seq of T WT embryos in vivo at E8.25. While Tal1 is expressed, previous reports have shown that it binds to the Lmo2 −75 enhancer in mesodermal cells, and not the Lmo2 −70 enhancer. *ChIP‐Seq tracks from 45 can be found in Fig EV5C. The only factor that is expressed and bound at the Lmo2‐70 enhancer by ChIP‐Seq is T.
  6. WISH analysis of Lmo2, Tal1, and T expression in control T WT compared to Lmo2‐70 ΔTBS/ΔTBS embryos from E8.5 to E9.5. Black dotted lines indicate the magnification of a lateral view of the caudal end of the embryo, shown in the inset. The second inset pictured below is a dorsocaudal view of the tail. White arrowheads indicate Lmo2 expression in the caudal end mesoderm. Black arrowheads indicate Lmo2 expression in the intersomitic vessels. Scale bar: 500 μm.
  7. RT‐qPCR analysis of gene expression in the caudal ends of E8.25 Lmo2‐70 ΔTBS/ΔTBS embryos compared to T WT control. The mean of n = 2 biological replicates of pools of five microdissected caudal ends is depicted.