Heat shock factor sites in Alu repeats are functionally active. Reporter constructs of the promoter region of three genes, SPN (G1), SPINK6 (G2) and HIST1H4A (G3), containing a HSF site within Alus when cloned downstream of a minimal promoter containing a firefly luciferase construct show more than two-fold induction in response to heat shock. Site-directed mutagenesis (SDM) in the predicted HSF within Alus led to reduced induction of approximately 1.4-fold in response to heat shock stress. Data were normalized using co-transfected renilla luciferase vector. The experiment was repeated three times and in triplicates to analyze standard deviations in all cases. HSPA1A was used as a positive control after an earlier published report of the functional HSF in the promoter region of the gene; the HSF site is in a non-Alu region of the HSPA1A gene promoter. G1, G2 and G3 represent wild type (WT) constructs, whereas G1*, G2* and G3* represent the SDM constructs. The statistical significance (Student's t-test) of the observed expression changes in response to heat shock in WT and SDM constructs as well as between them are shown in the lower panel, where figures in bold represent a significant P-value change. It is worth mentioning that the SDM constructs do not show significant expression changes following heat shock whereas WT and WT versus SDM show significant changes in expression. Error bars represent standard deviation among experimental replicates.