NPAT activates histone H4 transcription. (A) NPAT activates transcription from H4 promoters. U2OS cells were transfected with 1 μg pCMV (vector) or 1 μg pCMV–NPAT together with 50 ng pGLH4 (labeled as H4), pGLH4-1 (labeled as H4-1), pGL3 control (labeled as Control), pGL–b-bmyb (labeled as b-myb), or pGL–dhfr (labeled as dhfr) reporter construct. Fifty ng pCMV–lacZ was also co-transfected in each transfection. Thirty-six hours after transfection, cells were lysed and the activities of β-galactosidase and luciferase were assayed as described in Materials and Methods. The activities of the β-galactosidase were used to normalize the transfection efficiency among different samples. Fold of induction was calculated by comparing the luciferase activity from NPAT-transfected cells with that from the vector transfected cells. The figure shows the mean results and standard deviations from at least three independent experiments. (B) U2OS cells were transfected with 1 μg pCMV together with 50 ng pGLH4, pGLH4(80), pGLH4(65), pGLH4(40), pGLH4(D1), or pGLH4(M1) (as indicated as H4, 80, 65, 40, D1, and M1, respectively). Thirty-six hours after transfection, the cells were lysed and the β-galctosidase and luciferase activities were measured as described in A. The data represent the results from at least three independent experiments. (C) Activation of histone H4 transcription by NPAT is dependent on the H4 SSCS. U2OS cells were transfected with 1 μg vector or 1 μg pCMV–NPAT together with 50 ng of indicated reporter construct. Thirty-six hours after transfection, the samples were analyzed as described in A. H4, 80, 65,40, D1, and M1 represent the plasmids as described in B. The means and standard deviations from at least three independent experiments are depicted.
