Figure 3. Variant hair follicle enhancers produce altered levels of gene expression.

(a–c) Representative E16.5 transgenic embryos, generated by pronuclear injection of different 6.7 kb H2-lacZ constructs (shown below), processed for lacZ gene activity (blue). The full H2 region was used for these experiments, as expression in kidney provided a control for successful integration and expression of constructs even if expression in hair was disrupted. The clones tested were: (a) H2-ANC, "A" at rs12821256, (b) H2-BLD, "G" at rs12821256, or (c) H2-DEL, an 11 bp deletion that removes the rs12821256 position. lacZ gene activity was observed in developing hair follicles and kidneys (arrows) in all transgenic embryos. Although no consistent difference was noted between H2-ANC (N=15) and H2-BLD (N=9) embryos, H2-DEL embryos (N=8) (c) showed reduced activity in skin but normal kidney expression (arrow). Scale bar, 1 mm. (d) Expression analysis of different 1.9 kb HFE-luciferase reporters in the human HaCaT keratinocyte cell line. Bars represent the mean increase in luciferase gene activity over an empty vector control measured 48 hours after transfection from a typical experiment. The enhancers tested differed only by the following: HFEANC (A at rs12821256), HFE-BLD (G at rs12821256), and HFE-DEL (11 bp deletion removing rs12821256). Both the HFE-BLD and HFE-DEL constructs exhibited significantly reduced activity in HaCaT keratinocytes compared to the HFE-ANC plasmid. Error bars indicate s.e.m. Unpaired t test P-values; * P<0.05, *** P<5×10⁻⁴.