The Cyclin E/Cdk2 Substrate and Cajal Body Component p220^NPATActivates Histone Transcription through a Novel LisH-Like Domain

Abstract

p220NPAT 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 G 1 , S, and G 2 phases and two additional Cajal bodies tethered to histone genes at 1q21 during S, and G 2 phases. Overexpression of p220 in U2OS cells can promote the G 1 /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 ϳ30-aminoacid 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.Cell cycle transitions are regulated by cyclin-dependent kinases, a family of enzymes composed of a catalytic Cdk subunit and a regulatory cyclin subunit (19). Cdk proteins act to promote specific cell cycle events, such as DNA replication and entry into mitosis, by phosphorylating particular substrates. E-, D-, and A-type cyclins function together with Cdk4 and Cdk2 to promote entry into and completion of DNA synthesis (19). Important targets for these kinases include a family of transcriptional repressors typified by the retinoblastoma protein.Phosphorylation of Rb leads to derepression of E2F transcription factors, which then activate a transcriptional program designed to promote S phase (reviewed in references 14 and 26).DNA synthesis is accompanied by additional transcriptional programs that promote completion of replication. One of these is the activation of core histone synthesis that occurs as cells enter S phase. Histones are the building blocks of nucleosomes and are required for packaging of newly synthesized DNA into chromatin. Histone synthesis is complex and is regulated by multiple mechanisms which together provide an ϳ35-fold increase in the quantity of histone mRNA during S phase (20; for a review, see reference 21). Histone gene transcription is activated ϳ5-fold as cells enter S phase (20). Linker histone (H1) and core histones (H2A...

Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland

“…Cyclin D expression appears to be responsive to mitogenic stimuli and therefore gain-of-function mutations, including gene amplification, may represent a bypass of various receptor/ligand-dependent mitogenic stimuli. Cyclin E functions downstream of cyclin D and appears to contribute to entry into S-phase both through the hyperphosphorylation of Rb and by Rb-independent mechanisms (Lukas et al, 1997;Seghezzi et al, 1998;Geisen and Moroy, 2002;Wei et al, 2003).…”

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220NPAT in human embryonic stem cells

“…One principal event during the somatic cell cycle is the induction of the HiNF-P/ p220 NPAT co-activation complex by CDK dependent phosphorylation of p220 NPAT (Zhao et al, 1998(Zhao et al, , 2000Ma et al, 2000;Mitra et al, 2003;Wei et al, 2003;Ye et al, 2003;Miele et al, 2005). Here we examine the appearance of subnuclear foci containing p220 NPAT and phosphorylation of p220 NPAT in ES cells to understand the regulation of the G1/S phase transition relative to somatic cells.…”

“…Phosphorylation of p220 NPAT at CDK-related epitopes was examined using specific antibodies that recognize two different regions within p220 NPAT (i.e., P-T1270 or P-T1350) Wei et al, 2003). Cyclin staining was carried out with rabbit polyclonal cyclin A (H-432) (1:200; Santa Cruz Biotechnology), rabbit polyclonal cyclin B1 (H-433) (1:200; Santa Cruz Biotechnology), and rabbit polyclonal cyclin E (C-19) (1:200; Santa Cruz Biotechnology).…”

“…Similar to somatic cells, ES cells express histone gene regulatory factors (e.g., HiNF-P and p220 NPAT ) and exhibit S phase specific expression of distinct members of the DNA replication dependent histone gene family (Becker et al, 2006(Becker et al, , 2007. In somatic cells, critical events for the onset of S phase include the cell cycle dependent phosphorylation of p220 NPAT by cyclin E/CDK2 at nuclear foci related to Cajal bodies and the subsequent recruitment of p220 NPAT by transcription factor HiNF-P to histone H4 gene promoters (Zhao et al, 1998(Zhao et al, , 2000Ma et al, 2000;Mitra et al, 2003;Wei et al, 2003;Ye et al, 2003;Miele et al, 2005).…”

FLASH is required for histone transcription and S-phase progression

“…In addition to participating in various RNA-processing activities, CBs have also been implicated in transcriptional regulation of the cell-cycledependent histone genes. Phosphorylation of a CB component p220͞nuclear protein, ataxia-telangiectasia (NPAT) by cyclin E͞Cdk2 is required for activation of histone transcription, exit from G 1 , and progression through S phase (7)(8)(9)(10)(11)(12). Taken together, these observations suggest that CBs are intimately involved in histone gene expression.…”