The Cyclin E/Cdk2 Substrate p220 NPAT Is Required for S-Phase Entry, Histone Gene Expression, and Cajal Body Maintenance in Human Somatic Cells

Abstract

Cyclin E/Cdk2, a central regulator of the G1/S transition, coordinates multiple cell cycle events, including DNA replication, centrosome duplication, and activation of the E2F transcriptional program. Recent studies suggest a role for cyclin E/Cdk2 in activation of histone transcription during S phase via the Cajal body-associated protein p220NPAT, and in addition, p220 can promote S-phase entry independently of histone transcriptional activation when overexpressed. Here we have examined the requirement for p220 in histone transcription, cell cycle progression, and Cajal body function through analysis of human somatic HCT116 cells engineered to contain a conditional p220 allele. p220 is required for proliferation of HCT116 cells, as assessed after expression of Cre recombinase in p220(flox/-) cells. This defect was due to an inability of these cells to transit from G0/G1 into S phase, and cell cycle arrest occurred in the presence of elevated Cdk2 kinase activity. Expression of human papillomavirus E7, but not E6, eliminated cell cycle arrest in response to p220 depletion. Optimal expression of all four core histone genes required p220, as did optimal transcription of a histone H4 promoter-luciferase construct. Basal histone H4 expression in G0/G1, although p220 dependent, occurs in the absence of detectable phosphorylation of p220 on Cdk2 sites. Cells lacking p220 displayed defects in the localization of the Cajal body component p80coilin as cells progressed from G0 to S phase in response to mitogenic signals. These finding indicate that p220 is an essential downstream component of the cyclin E/Cdk2 signaling pathway and functions to coordinate multiple elements of the G1/S transition.