Chromatin remodeling is required for efficient transcription of eukaryotic genes. In a genetic selection for budding yeast mutants that were defective in induction of the phosphate-responsive PHO5 gene, we identified mutations in ARG82/IPK2, which encodes a nuclear inositol polyphosphate kinase. In arg82 mutant strains, remodeling of PHO5 promoter chromatin is impaired, and the adenosine triphosphate-dependent chromatin-remodeling complexes SWI/SNF and INO80 are not efficiently recruited to phosphate-responsive promoters. These results suggest a role for the small molecule inositol polyphosphate in the regulation of chromatin remodeling and transcription.DNA in the eukaryotic nucleus is packaged into chromatin, which forms a repressive structure that tends to limit the access of DNA-binding proteins to DNA. Cellular activities have been identified that function to counteract chromatin-mediated repression through acetylation, methylation, or phosphorylation of histones (1). Additionally, complexes such as SWI/SNF alter the association of histones with DNA by using the energy from adenosine triphosphate (ATP) hydrolysis (2). Though many chromatin-modifying activities have been characterized mechanistically, little is known about their regulation.The budding yeast PHO5 promoter and gene compose a useful system to investigate the relation between chromatin structure and gene expression. Transcription of PHO5 is regulated in response to phosphate availability by the transcription factors Pho4 and Pho2 (3). When yeast cells are grown in a phosphate-rich medium, Pho4 is phosphorylated by the cyclin-CDK (cyclin-dependent kinase) complex Pho80-Pho85 (4) and inactivated (5). In addition, four positioned nucleosomes reside over the PHO5 promoter, and PHO5 transcription is repressed (6). Upon phosphate starvation, Pho4 is unphosphorylated and active (5), the positioned nucleosomes are no longer detectable (6), and PHO5 is induced. Remodeling of PHO5 chromatin structure requires Pho4 and Pho2 (7) and is facilitated by the histone acetyltransferase Gcn5, which acetylates histones in the promoter region (8,9).To identify additional factors important for remodeling chromatin at the PHO5 promoter, we designed a genetic selection to identify mutants defective in PHO5 transcription [Supporting Online Material (SOM) Text]. This selection identified mutations in PSE1, which encodes the import receptor for Pho4 (10), and a mutation in ARG82/IPK2 (denoted arg82-153) (SOM Text). Under inducing conditions, PHO5 transcription and chromatin remodeling are reduced in the arg82-153 mutant ( fig. S1).
Inositol phosphates are a group of highly complex molecules for which cellular functions are being defined; however, this process has been difficult because of the large number of different inositol phosphates, with varying numbers of phosphates in different positions of the inositol ring. The synthetic pathway generating higher order inositol phosphates, inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P 5 ) 1 and inositol hexakisphosphate (InsP 6 ), in human cells is not completely defined. Current evidence supports inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) as the common precursor and Ins(1,3,4,5,6)P 5 and InsP 6 as the predominant higher inositol phosphates in human cells, and the synthetic pathway from Ins(1,4,5)P 3 to Ins(1,3,4,5,6)P 5 remains an area of active investigation (for general reviews, see Refs. 1 and 2). Hence, the goal of the current study is to define a catalytic step leading to Ins(1,3,4,5,6)P 5 synthesis.The best characterized InsP 6 synthetic pathway is in Saccharomyces cerevisiae (3). The common precursor Ins(1,4,5)P 3 is synthesized from phospholipase C and subsequently used by two distinct proteins, Ipk2p and Ipk1p, ultimately to generate InsP 6 . The IPK1 and IPK2 genes were isolated from a genetic screen for mutants that were synthetically lethal in combination with a gle1-2 mutant, which is defective in mRNA export. Sequential phosphorylation of Ins(1,4,5)P 3 at the D-6 and D-3 positions generates Ins(1,4,5,6)P 4 and Ins(1,3,4,5,6)P 5 , respectively, catalyzed by the IPK2 gene product (4). Next, phosphorylation at the D-2 position yields InsP 6 catalyzed by the IPK1 gene product (5).In human cells, the synthetic pathway to higher inositol phosphates shares the same initial step to produce the common precursor Ins(1,4,5)P 3 catalyzed by phospholipase C (2). However, the pathway from Ins(1,4,5)P 3 to Ins(1,3,4,5,6)P 5 is less clear. Two different synthetic pathways generating different isomers of InsP 4 have been described. First, the synthesis of Ins(1,3,4,5)P 4 from Ins(1,4,5)P 3 has been well documented to be catalyzed by inositol 1,4,5-trisphosphate 3-kinases (InsP 3 3-kinases) in response to stimulation by various ligands (6, 7). Genes encoding the InsP 3 3-kinase have been cloned (8 -10) and the enzymatic catalysis characterized (6). Second, the synthesis of Ins(1,3,4,6)P 4 from Ins(1,3,4)P 3 was described to be catalyzed by the inositol 1,3,4-trisphosphate 5/6-kinase (InsP 3 5/6-kinase), which has no activity toward the Ins(1,4,5)P 3 isomer (11). The Ins(1,3,4)P 3 isomer is synthesized from Ins(1,3,4,5)P 4 catalyzed by 5-phosphatase(s). The human enzymes responsible for the conversion of either Ins(1,3,4,5)P 4 or Ins(1,3,4,6)P 4 to Ins(1,3,4,5,6)P 5 have not been reported. The final step is the phosphorylation of Ins(1,3,4,5,6)P 5 at the D-2 position catalyzed by inositol 1,3,4,5,6-pentakisphosphate 2-kinase (12).To date, the synthesis of Ins(1,3,4,5,6)P 5 from InsP 4 has been characterized in S. cerevisiae, as noted above, and in rat. The rat inositol phosphate multikinas...
Production of inositol hexakisphosphate (IP 6 ) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP 6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116⌬ipk1⌬ and nup42⌬ipk1⌬ double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP 6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP 6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP 6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP 6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP 6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP 6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP 6 production is sufficient for mediating the Gle1-mRNA export pathway.The nucleus is the defining structure of a eukaryotic cell and houses the genetic information that characterizes the organism. The nuclear compartment is separated from the cytoplasm by the nuclear envelope (NE), 1 two lipid bilayer membranes that join to form pores containing nuclear pore complexes (NPCs). The 60-MDa NPC structure is assembled from multiple copies of ϳ30 distinct proteins designated nucleoporins (Nups) (1, 2). The central region of the NPC consists of inner and outer rings connected by a series of spokes. Fibrils extend from either face of the NPC, and the filaments on the nuclear side are joined at the distal end to form a basket structure (3). The NPCs create aqueous semipermeable portals across the NE that allow the passive diffusion of small molecules. However, translocation of macromolecules through the NPC requires facilitated transport mediated by Nups, other NPC-associated proteins, and shuttling transport factors (4 -6). One class of macromolecules that must traverse the NPC is mRNAs. The movement of mRNA through the NPC is a regulated process in the progression from DNA transcription in the nucleus to the production of a translated protein in the cytoplasm (7,8). To ensure proper gene expression, there are...
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