Abstract
The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described. We have determined, by real-time quantitative PCR and RNase protection, that the human histone H4 genes are not equally expressed and that a subset contributes disproportionately to the total pool of H4 mRNA. Differences in histone H4 gene expression can be attributed to observed unequal activities of the H4 gene promoters, which exhibit variations in gene regulatory elements. The overall expression pattern of the histone H4 gene complement is similar in normal and cancer cells. However, H4 genes that are moderately expressed in normal cells are sporadically silenced in tumor cells with compensation of expression by other H4 gene copies. Chromatin immunoprecipitation analyses and in vitro DNA binding assays indicated that 11 of the 15 histone H4 genes interact with the cell cycle regulatory histone nuclear factor P, which forms a complex with the cyclin E/CDK2-responsive co-regulator p220(NPAT). These 11 H4 genes account for 95% of the histone H4 mRNA pool. We conclude that the cyclin E/CDK2/p220(NPAT)/histone nuclear factor P signaling pathway is the principal regulator of histone H4 biosynthesis.
Highlights
Histones have crucial roles in replication, transcription, repair, and recombination [1,2,3]
The relative contributions of individual histone H4 genes to the total H4 mRNA pool were determined with primer pairs specific to the unique 5Ј-untranslated region of each H4 gene (TABLE TWO)
The mRNA levels of each histone H4 gene were up-regulated in S phase between 5- and 20-fold above the levels in G1 and G2 phases
Summary
HiNF, histone nuclear factor; qPCR, quantitative PCR; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative reverse transcription-PCR; RPAs, RNase protection assays; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay. A Albig, W., Kioschis, P., Poustka, A., Meergans, K., and Doenecke, D. B Albig, W., Kardalinou, E., Drabent, B., Zimmer, A., and Doenecke, D. E Sierra, F., Stein, G., and Stein, J. We used quantitative PCR (qPCR) and chromatin immunoprecipitation (ChIP) to examine the expression and regulation of the 15 human histone H4 genes in normal and tumor-derived cells. Our results firmly establish that 11 genes, which account for Ͼ95% of histone H4 mRNAs, are coordinately controlled during the cell cycle and are responsive to the cyclin E/CDK2/p220NPAT/HiNF-P signaling pathway
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