Fig. 8.

p57^Kip2 suppresses histone H4 promoter activity in normal diploid human cells. (A) Total RNA from wild type p57 (WT), heterozygous p57 null (HET) and homozygous p57 null (KO) mouse embryonic fibroblasts was examined for mRNA expression of mouse HiNF-P, Hist2H4, HistH4/m, Hist1H4/f, p57, p27 and p21 using quantitative RT-PCR. Values were calculated with the ΔΔCT method using HPRT as an internal control. We did not observe significant changes in the expression of Hist2H4, Hist1H4/m and Hist1H4/f histone in HET and KO cells compared to WT cells. As expected p57 mRNA is absent in KO and HET cells (HET cells are functionally null for p57 due to imprinting), while expression of p21 mRNA in KO and HET cells is significantly higher than in WT cells. Student’s t-test (unpaired, 2-tailed, non-parametric) was used to calculate significance by comparing gene expression in WT to either HET or KO cells. Asterisks (*) indicate P-values <0.05). (B) WI-38 human diploid fibroblasts were plated at a density of 1.6×10⁵/well in six-well plates and transiently transfected at day 2 after plating at a cell density of ~30% with wild-type histone H4 promoter luciferase reporter construct. Cells were co-transfected with the indicated amounts of expression vectors for HiNF-P (P), p220^NPAT (N) and p57, or an empty vector (EV). Experiments were performed with either 200 ng (left graph) or 600 ng (right graph) of firefly luciferase reporter gene construct. The promoterless Renilla luciferase control plasmid was also different (25 ng, left graph; 75 ng, right graph), but total amount of DNA was maintained at 2.5 μg in every transfection.