The highly homologous Rnf2 (Ring1b) and Ring1 (Ring1a) proteins were identified as in vivo interactors of the Polycomb Group (PcG) protein Bmi1. Functional ablation of Rnf2 results in gastrulation arrest, in contrast to relatively mild phenotypes in most other PcG gene null mutants belonging to the same functional group, among which is Ring1. Developmental defects occur in both embryonic and extraembryonic tissues during gastrulation. The early lethal phenotype is reminiscent of that of the PcG-gene knockouts Eed and Ezh2, which belong to a separate functional PcG group and PcG protein complex. This finding indicates that these biochemically distinct PcG complexes are both required during early mouse development. In contrast to the strong skeletal transformation in Ring1 hemizygous mice, hemizygocity for Rnf2 does not affect vertebral identity. However, it does aggravate the cerebellar phenotype in a Bmi1 nullmutant background. Together, these results suggest that Rnf2 or Ring1-containing PcG complexes have minimal functional redundancy in specific tissues, despite overlap in expression patterns. We show that the early developmental arrest in Rnf2-null embryos is partially bypassed by genetic inactivation of the Cdkn2a (Ink4a͞ARF) locus. Importantly, this finding implicates Polycomb-mediated repression of the Cdkn2a locus in early murine development. P olycomb Group (PcG) proteins and their genetic counterparts, the trithorax Group proteins (trxG), maintain Hox gene expression boundaries (1-4), which are critical for regional patterning along the antero-posterior (AP) axis (5-7). Based on biochemical characteristics, mammalian PcG proteins are currently grouped into at least two distinct functional groups: the first comprises Eed, Ezh1, and Ezh2 in the mouse (8, 9); the second consists of the highly related protein pairs Cbx2 (M33)͞Cbx4 (MPc2), Bmi͞Zfp144 (Mel18), and Edr1 and 2 (Rae28͞Mph1 and 2), respectively (10, 11). For ease of this discussion we refer to them as groups I and II, respectively. Group I and II homologs are evolutionarily conserved from Drosophila to humans, only group I homologs are found in plants and Caenorhabditis elegans as well, supporting the concept of separate function (12, 13). In addition, complex composition differs throughout development in a temporal and cell-type-specific manner (14, 15). Interaction of Eed with histone deacetylases and the intrinsic histone methyltransferase activity of Ezh proteins suggest mechanisms for repression by group I complexes (16)(17)(18)(19). Although a mammalian hPRC-H (group II) complex harbors an intrinsic capacity to stabilize a repressive chromatin structure and counteract SWI͞SNF chromatin remodeling complexes in vitro, its in vivo mode of action is not well understood (20). Association with histone methyl transferase activity may in part help explain the repressive action of some group II complexes (21).Rnf2 and Ring1 have been identified as in vivo interactors of the group II PcG protein Bmi1 by us and others (22). These Ring finger proteins...

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane traf®cking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P 2 : phosphatidylinositol 4-phosphate 5-kinase a (Type Ia PIPkinase). We ®nd that both PLD1 and PLD2 interact with the Type Ia PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ia PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P 2 is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.

We describe for the first time a novel in vivo activity for Type Ialpha PIPkinase, and a novel pathway for the in vivo synthesis of functional PtdIns(3,4,5)P(3), a key lipid second messenger regulating a number of diverse cellular processes.

It is now clear that phosphoinositides, which play a major role in the regulation of a variety of cellular processes in the cytoplasm, are found within the nucleus. Their role in this subcellular compartment is still contentious: however, data has suggested that nuclear inositides generate substrates, such as PtdIns(4,5)P2, utilised by a number of nuclear signalling pathways: for example, nuclear phospholipase C and the PtdIns 3-kinase cascade. There is also evidence that PtdIns(4,5)P2 may play a role in the localisation and regulation of a number of nuclear proteins such as the BAF complex, which is involved in the regulation of chromatin structure. Although the presence of nuclear inositides has been demonstrated in a number of different cell types, suggesting that it is ubiquitous, there are many inconsistencies within the literature concerning the locations and isotypes of enzymes that are involved in their regulation and in the potential second messengers which are generated by them. This review aims to highlight some of these inconsistencies in order to focus on areas that need further characterisation.

Inositide signaling at the plasma membrane has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, vesicle trafficking, and gene transcription. Studies have also shown that a distinct inositide pathway exists in nuclei, where it may regulate nuclear processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. We previously demonstrated that nuclear PtdIns(4,5)P(2) synthesis is stimulated during progression from G1 through S phase, although mechanistic details of how cell cycle progression impinges on the regulation of nuclear inositides is unknown. In this study, we demonstrate that pRB, which regulates progression of cells from G1 through S phase interacts both in vitro and in vivo with Type I PIPkinases, the enzymes responsible for nuclear PtdIns(4,5)P(2) synthesis. Moreover, this interaction stimulates the activity of Type Ialpha PIPkinase in an in vitro assay. Using murine erythroleukamia (MEL) cells expressing a temperature-sensitive mutant of large T antigen (LTA), we demonstrate changes in vivo in nuclear PtdIns(4,5)P(2) levels that are consistent with the ability of LTA to disrupt pRB/Type I interactions. This study, for the first time, provides a potential mechanism for how cell cycle progression could regulate the levels of nuclear inositides.