T-ALL expression of <i>HHEX</i> requires its promoter and enhancer.

<p>(<b>A</b>) Schematic shows the structure of the mouse <i>Hhex</i> gene with 4 exons (coding sequences shown in blue). We analyzed ChIP-seq data from anti-Ldb1 that showed occupancy within intron 1 (green bar graph). The RefSeq mRNAs for <i>Hhex</i> are shown below the sequencing tag counts. The bar graph under this shows the conservation across multiple mammalian species. The red box denotes an enhancer that was previously functionally characterized as specifying blood-specific expression of <i>Hhex</i>. (<b>B</b>) We cloned the enhancer shown in panel A 5′ to the SV40 promoter and transfected it into LOUCY, K562, and Jurkat cell lines along with pCMV-<i>Renilla</i> luciferase vector. Luciferase values were normalized to <i>Renilla</i> to correct for transfection efficiency and expressed as fold over <i>Renilla</i> alone. (<b>C</b>) We constructed a luciferase expression vector with 1 kb of the <i>Hhex</i> promoter, exon 1, intron 1, and replaced exon 2 with the luciferase cDNA. We transfected this reporter (pG-<i>Hhex</i>-luc) into K562 and LOUCY cells. Clones 1–6 have 50–100 bp deletions 5′ to 3′ within the enhancer region. Luciferase expression was normalized to <i>Renilla</i> for transfection efficiency and expressed as fold above that seen in <i>Renilla</i> alone.