Figure 9.

Cyclin E–Cdk2 regulates NPAT-mediated activation of histone gene transcription. (A) Cyclin E–Cdk2 stimulates NPAT-mediated transcriptional activation. U2OS cells were transfected with 50 ng pGLH4(65) together with 50 ng pCMV–lacZ, and the indicated expression plasmids. 0.5 μg of each expression plasmid was used, and if needed, pCMV vector was added to bring the total amount of plasmid DNA to 1.6 μg. The samples were analyzed as described in Fig. 6A. (B) The stimulation of NPAT-mediated transcriptional activation by cyclin E–Cdk2 is the SSCS dependent. The experiments were carried out as described in A except that the reporter used was pGLH4 (M1). (C) Cyclin E–Cdk2 stimulates NPAT-mediated H4 transcriptional activation in non–S phase cells. U2Os cells, grown on 10-cm plates, were transfected with 300 ng pGLH4(65) and 300 ng pCMV–lacZ, together with indicated expression plasmids (NPAT, 6 μg; cyclin E and Cdk2, 1.5 μg). pCMV (vector) was added to bring the total plasmid DNA concentration to 9 μg/plate. Five hours after transfection, Nocadazole (final concentration 70 ng/mL) was added into the culture medium to arrest the cells in M phase. Cells in M phase were harvested by mitotic shake-off 18 h later. The cells were washed twice with culture medium and replated. One and one-half hours after replating, the effect of NPAT on H4 transcription was analyzed as described in Fig. 6A. The mean results from two independent experiments are shown. (D) Association of NPAT and cyclin E with histone gene clusters. Lysates prepared from U2OS cells were precipitated with the indicated antibodies. The indicated DNA sequences were detected by PCR using specific primers as described in Materials and Methods.