No abstract

The yeast nucleosome assembly protein 1 (yNap1) plays a role in chromatin maintenance by facilitating histone exchange as well as nucleosome assembly and disassembly. It has been suggested that yNap1 carries out these functions by regulating the concentration of free histones. Therefore, a quantitative understanding of yNap1-histone interactions also provides information on the thermodynamics of chromatin. We have developed quantitative methods to study the affinity of yNap1 for histones. We show that yNap1 binds H2A/H2B and H3/H4 histone complexes with low nM affinity, and that each yNap1 dimer binds two histone fold dimers. The yNap1 tails contribute synergistically to histone binding while the histone tails have a slightly repressive effect on binding. The (H3/H4) 2 tetramer binds DNA with higher affinity than it binds yNap1.Histone chaperones are a diverse group of acidic proteins that bind histones and participate in chromatin assembly and disassembly during replication and transcription. Many members of the various chaperone families play additional and often ill-described roles in cell cycle regulation, apoptosis, and DNA damage repair. Several histone chaperones exhibit tissue-specific functions in transcription regulation (recently reviewed in Refs. 1, 2). As the dynamic nature of nucleosomes and chromatin has become evident, the role of histone chaperones in the modulation of chromatin structure is increasingly recognized. However, little mechanistic insight into the processes of chaperone-mediated nucleosome assembly and disassembly is available. In particular, no quantitative information exists on histone binding, the most fundamental function of histone chaperones.Nucleosome assembly protein 1 (Nap1) 4 was one of the first histone chaperones to be identified (reviewed in Refs. 3, 4). The structure of yeast Nap1 (yNap1) reveals a novel fold that is likely conserved among other members of the Nap1 family (5). At low micromolar concentrations yNap1 exists as a homodimer (6, 7). The large dimer interface is predominantly hydrophobic in character and buries ϳ22% of the overall surface of yNap1 (5); it is therefore unlikely that yNap1 exists in a monomeric state except for perhaps under the most dilute conditions (6). All Nap1 family members have a C-terminal acidic domain (CTAD) of varying length that is not required for histone binding and chromatin assembly, and a variable N-terminal tail of unknown function (3).Nap1 binds all four core histones as well as the linker histone H1 (8 -10). Nap1-mediated nucleosome formation in vitro is characterized by the transfer of a (H3/H4) 2 tetramer onto DNA, followed by the incorporation of H2A/H2B dimer (8). Nakagawa et al. (11) have qualitatively shown that the affinity of the (H3/H4) 2 tetramer for DNA exceeds its affinity for Nap1, and that the affinity of H2A/H2B for a DNA-bound (H3/H4) 2 tetramer (a tetrasome) is greater than its affinity for Nap1.Our understanding of the thermodynamics of chaperonehistone interactions in general, and of Nap1-histone interact...

The increasing availability of personal genomic tests has led to discussions about the validity and utility of such tests and the balance of benefits and harms. A multidisciplinary workshop was convened by the National Institutes of Health and the Centers for Disease Control and Prevention to review the scientific foundation for using personal genomics in risk assessment and disease prevention and to develop recommendations for targeted research. The clinical validity and utility of personal genomics is a moving target with rapidly developing discoveries but little translation research to close the gap between discoveries and health impact. Workshop participants made recommendations in five domains: (1) developing and applying scientific standards for assessing personal genomic tests; (2) developing and applying a multidisciplinary research agenda, including observational studies and clinical trials to fill knowledge gaps in clinical validity and utility; (3) enhancing credible knowledge synthesis and information dissemination to clinicians and consumers; (4) linking scientific findings to evidence-based recommendations for use of personal genomics; and (5) assessing how the concept of personal utility can affect health benefits, costs, and risks by developing appropriate metrics for evaluation. To fulfill the promise of personal genomics, a rigorous multidisciplinary research agenda is needed.

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 4-kinases is the first step in the synthesis of polyphosphoinositides, the lipid precursors of intracellular signaling molecules. We have recently identified a cytosolic PtdIns 4-kinase (cPI4K) in the bovine adrenal cortex that is distinguished from previously known PtdIns 4-kinases by its sensitivity to the PtdIns 3-kinase inhibitor wortmannin (WT). The present study has further characterized this soluble enzyme and compared its properties to those of the membrane-bound, type II PtdIns 4-kinase activity of the adrenal cortex and the type III enzyme of bovine brain. The enzymatic activity of adrenal cPI4K was inhibited not only by WT (IC50 approximately 50 nM) but also by LY-294002 (IC50 approximately 100 microM), another inhibitor of PtdIns 3-kinase, and neither compound affected type II PtdIns 4-kinase at concentrations that inhibited cPI4K. In contrast to the type II enzyme, cPI4K had a significantly higher Km for ATP, was relatively insensitive to inhibition by adenosine (Ki approximately 800 microM vs approximately 40 microM), had lower affinity for PtdIns, and was not inhibited by Ca2+ ions. These properties identify the WT-sensitive adrenal cPI4K as a type III PtdIns 4-kinase that is distinct from the tightly membrane-bound, Ca2+- and adenosine-sensitive, type II PtdIns 4-kinase. The type III PtdIns 4-kinase prepared from bovine brain exhibited similar kinetic parameters as the adrenal cPI4K, and was also inhibited by WT with an IC50 of 30-50 nM. Since WT inhibits the synthesis of agonist-regulated phosphoinositide pools in intact cells at micromolar concentrations, these findings indicated that type III rather than type II PtdIns 4-kinases are responsible for the maintenance of the precursor phospholipids required for intracellular signaling through the inositol phosphate/Ca2+ pathway.