Enhancer mapping identifies putative transcription factor binding sites necessary for enhancing FSHB transcription. (A) Luciferase expression after transfection into LβT2 cells from subregions (halves or quarters) of the human 450 bp enhancer subcloned upstream of the human −1028/+7 FSHB promoter. A subregion spanning 216-341 was sufficient for enhancer function (n = 3). (B) Luciferase expression from the human 216-341 subregion cloned in the reverse orientation (RV 216-341) compared with the forward orientation (FW 216-341) and human −1028/+7 FSHB promoter alone (n = 5). (C) From the 450-base-pair enhancer subcloned upstream of the human −1028/+7 FSHB promoter, a series of constructs were created with 10-base-pair deletions spanning 216-345. Δ indicates the span of the 10-base-pair deletion in each construct. Expression levels from four deletion constructs were significantly different from the full-length construct (n = 5). Putative transcription factor binding sites were identified from the TRANSFAC database using the Match program and each consensus sequence is shown compared with the mouse and human genomic sequences at the corresponding region. The consensus motif is underlined in each genomic sequence. Bases that differ from the consensus are bolded. Luciferase values were normalized to the β-galactosidase internal control and are expressed relative to the empty reporter vector. Values represent mean ± SEM. Data were analyzed by one-way ANOVA, post hoc Dunnett multiple comparisons test. A square transform was used on data from A and a log transform on B prior to statistical analysis (* P < 0.05, *** P < 0.005). Abbreviation: P, promoter.