The Cyclin E/Cdk2 Substrate and Cajal Body Component p220NPATActivates Histone Transcription through a Novel LisH-Like Domain

Abstract

P220^NPAT is a substrate of cyclin E/Cdk2 that localizes in nuclear organelles called Cajal bodies in a cell cycle-regulated manner. In normal diploid fibroblasts, p220 is concentrated in two Cajal bodies tethered to histone gene clusters at chromosome 6p21 during G₁, S, and G₂ phases and two additional Cajal bodies tethered to histone genes at 1q21 during S, and G₂ phases. Overexpression of p220 in U2OS cells can promote the G₁/S transition and can also promote transcription from histone H2B and H4 luciferase reporter constructs. How p220 expression induces these activities and whether the two activities are related are unknown. In this study, we developed a “lox-scanning” mutagenesis approach to identify functional domains in p220. We identified two distinct functional regions of p220. The C-terminal half of the protein contains multiple elements that are required for its ability to induce S phase in transfected cells. In contrast, sequences at the N terminus appear to be critical for activation of histone H4 and H2B reporter constructs. We identified an ∼30-amino-acid motif at the N terminus of p220 that has the characteristics of a LisH motif. LisH motifs are found in a large number of proteins in the database but are of unknown function. Point mutations in conserved residues in the LisH motif of p220 block histone H4 transcriptional activity without affecting localization in Cajal bodies or phosphorylation on Cdk2 phosphorylation sites. These studies indicate that the ability of p220 to promote S phase is independent of its ability to promote histone H4 transcription and suggests that p220 may link cyclin E/Cdk2 to multiple independent downstream functions.