Cell cycle-regulated phosphorylation of p220(NPAT) by cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription

Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcri ptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localiz ation of the cyclin E/Cdk2 interacting protein p220(NPAT) and its regulatio n by phosphorylation. p220 is localized to discrete nuclear foci. Diploid f ibroblasts in Go and G1 contain two p220 foci, whereas S- and G2-phase cell s contain primarily four p220 foci. Cells in metaphase and telophase have n o detectable focus, p220 foci contain cyclin E and are coincident with Caja l bodies (CBs), subnuclear organelles that associate with histone gene clus ters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromo some 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-speci fic antibodies against two of these sites react with p220 within CBs in a c ell cycle-specific manner. The timing of p220 phosphorylation correlates wi th the appearance of cyclin E in CBs at the G1/S boundary, and this phospho rylation is maintained until prophase. Expression of p220 activates transcr iption of the histone H2B promoter. Importantly, mutation of Cdk2 phosphory lation sites to alanine abrogates the ability of p220 to activate the histo ne H2B promoter. Collectively, these results strongly suggest that p220(NPA T) links cyclical cyclin E/Cdk2 kinase activity to replication-dependent hi stone gene transcription.