Heterochromatin is a structurally compacted region of chromosomes in which transcription and recombination are inactivated. DNA replication is temporally regulated in heterochromatin, but the molecular mechanism for regulation has not been elucidated. Among heterochromatin loci in Schizosaccharomyces pombe, the pericentromeric region and the silent mating-type (mat) locus replicate in early S phase, whereas the sub-telomeric region does not, suggesting complex mechanisms for regulation of replication in heterochromatic regions. Here, we show that Swi6, an S. pombe counterpart of heterochromatin protein 1 (HP1), is required for early replication of the pericentromeric region and the mat locus. Origin-loading of Sld3, which depends on Dfp1/Dbf4-dependent kinase Cdc7 (DDK), is stimulated by Swi6. An HP1-binding motif within Dfp1 is required for interaction with Swi6 in vitro and for early replication of the pericentromeric region and mat locus. Tethering of Dfp1 to the pericentromeric region and mat locus in swi6-deficient cells restores early replication of these loci. Our results show that a heterochromatic protein positively regulates initiation of replication in silenced chromatin by interacting with an essential kinase.

The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process.

Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system.

DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2 0 -deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as earlyfiring origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.

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To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.[Keywords: Cdc7-Drf1; DNA replication; Xenopus; cohesin; cohesion; Scc2-Scc4] Supplemental material is available at http://www.genesdev.org. Chromosomal DNA is duplicated in the S phase of the cell cycle and distributed to daughter cells in mitosis. Cohesion of sister chromatids is crucial for precise segregation of chromosomes, as it marks sister chromosomes to be separated. Cohesion is established by the ring-shaped cohesin complex, which consists of two structural maintenance of chromosome (SMC) family proteins, Smc1 and Smc3, an ␣-kleisin subunit, Rad21/Scc1, and an accessory protein, SA/Scc3 (for review, see Huang et al. 2005;Nasmyth and Haering 2005;Hirano 2006;Losada 2007). Several lines of evidence suggest that cohesion is established when replicated sister chromatids are both entrapped within the cohesin ring Nasmyth 2005, 2007), but alternative models also exist (Milutinovich et al. 2007). Proteolytic cleavage of Scc1 by separase at the metaphase-anaphase transition releases cohesins from chromatin, enabling sister chromatids to move to each daughter cell (Uhlmann et al. 2000). In metazoans, cohesins associated with the chromosome arms are removed in prophase by phosphorylation of the SA subunit via polo-like kinase (Sumara et al. 2000;Waizenegger et al. 2000;Losada et al. 2002;Hauf et al. 2005). The remainder of chromosome-linked cohesins is cleaved at the metaphase-anaphase transition by separase.The process of cohesion establishment is intimately connected to DNA replication. Experiments in yeast show that the establishment of cohesion in S phase requires that cohesin be present on chromatin during DNA replication (Uhlmann and Nasmyth 1998; Lengronne et al. 2006). The Ctf7/Eco1 protein, which physically and genetically interacts with PCNA and RFC (Kenna and Skibbens 2003;Moldovan et al. 2006), is required for establishment of cohesion in S phase (Skibbens et al. 1999;Toth et al. 1999)....

Insulin plays a key role in the stimulation of glucose uptake in tissues, such as muscle and adipocytes, as well as in the maintenance of glucose homeostasis. Impairment of insulin's ability to stimulate glucose uptake in the tissues is a major factor responsible for insulin resistance associated with type 2 diabetes.1) The primary mechanism of insulinstimulated glucose uptake in muscle and adipocytes is through the translocation of glucose transporter 4 (GLUT4) from intracellular pools to the plasma membrane.2) Insulin signal transduction is initiated by binding to the insulin receptor, followed by activation of the receptor tyrosine kinase (RTK).3,4) The activated RTK induces activation of downstream signaling pathways, such as phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The translocation of GLUT4 to the plasma membrane was established to be mediated through the PI3K pathway, based on the use of pharmacological inhibitors, and expression of a dominant negative mutant or constitutively active form of PI3K. [5][6][7][8][9] Flavonoids, which are primarily phenylbenzo-g-pyrone (phenylchromone) derivatives, are polyphenolic compounds present in fruits, vegetables, and beverages.10) It has been reported that flavonoids possess a variety of biological activities, including anti-inflammatory, anti-oxidant, anti-bacterial, anti-cardiovascular disease, and anti-cancer activities. 11)These actions were suggested to result from changes in the activity of a number of intracellular enzymes, including tyrosine kinases, protein kinase C, PI3K, and MAPK. [11][12][13][14] The findings suggest that flavonoids may modify insulin-stimulated glucose uptake by modulating insulin RTK and/or PI3K activity in muscle or adipose cells. Indeed, several reports have shown that flavonoids, such as myricetin, quercetin, catechin-gallate, genistein, and naringenin, inhibited insulinstimulated glucose uptake in adipocytes. [15][16][17] However, the inhibitory mechanisms may be different for flavonoids of different classes or structures. Bazuine et al. 15) reported that genistein, an isoflavone, directly inhibited insulin-stimulated glucose uptake in mouse 3T3-L1 adipocytes. On the other hand, naringenin, a flavanone, inhibited insulin-stimulated glucose uptake through blocking PI3K activity in the cell line.17) Therefore, in this study, we investigated the effects of flavonoids on insulin-induced glucose uptake in mouse MC3T3-G2/PA6 adipocytes using a panel of 24 flavonoids, including flavones, flavonols, isoflavones, flavanones, flavanols and flavanonols. MATERIALS AND METHODS MaterialsThe flavones, flavone, apigenin, luteolin and chrysin, the flavonols, kaempferol, quercetin, rutin and morin, the isoflavones, daidzein and genistein, the flavanones, hesperetin, hesperidin and naringenin, the flavanonol, silybin, the flavanols, (ϩ)-catechin and EGCG, fetal bovine serum (FBS) and insulin were purchased from SigmaAldrich Corp. (St. Louis, MO, U.S.A.). The flavone, baicalein, and the flavonols, gal...