Search

Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system., Howard Hughes Medical Institute, National Cancer Institute (U.S.) (Grant CA13106)

Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with skeletal muscle differentiation. One major subgroup of RMS tumors (so-called "fusion-positive" tumors) carries exclusive chromosomal translocations that join the DNA-binding domain of the PAX3 or PAX7 gene to the transactivation domain of the FOXO1 (previously known as FKHR) gene. Fusion-negative RMS represents a heterogeneous spectrum of tumors with frequent RAS pathway activation. Overtly metastatic disease at diagnosis is more frequently found in individuals with fusion-positive than in those with fusion-negative tumors. RMS is the most common pediatric soft-tissue sarcoma, and approximately 60% of all children and adolescents diagnosed with RMS are cured by currently available multimodal therapies. However, a curative outcome is achieved in <30% of high-risk individuals with RMS, including all those diagnosed as adults, those diagnosed with fusion-positive tumors during childhood (including metastatic and nonmetastatic tumors), and those diagnosed with metastatic disease during childhood (including fusion-positive and fusion-negative tumors). This white paper outlines current challenges in RMS research and their implications for developing more effective therapies. Urgent clinical problems include local control, systemic disease, need for improved risk stratification, and characterization of differences in disease course in children and adults. Biological challenges include definition of the cellular functions of PAX-FOXO1 fusion proteins, clarification of disease heterogeneity, elucidation of the cellular origins of RMS, delineation of the tumor microenvironment, and identification of means for rational selection and testing of new combination therapies. To streamline future therapeutic developments, it will be critical to improve access to fresh tumor tissue for research purposes, consider alternative trial designs to optimize early clinical testing of candidate drugs

Telomere-associated position-effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counterselected with 5-fluoroorotic acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV.

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P–3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.

In domestic disaster response, the affected state's National Guard frequently provides Civil Support to local civilian authorities. In some domestic catastrophes, the federal military may be asked to augment the National Guard response. It is in the context of Civil Support operations involving both the National Guard and federal military forces, that this paper examines the sometimes contentious C2 relationship between state and federal military forces. The thesis for discussion is that current domestic Civil Support C2, involving both Title 10 (federal military) and Title 32 (state National Guard) forces, can be improved by changes to existing law, directives and operating procedures. These modifications would allow the use of existing Joint C2 doctrine in such operations, creating Unity of Command with National Guard in the lead. The topic of modifying Civil Support C2 is not new. However, this paper contends that cultural resistance is a major factor in the inability of the Active Component to support change to C2. A change in the Title 10 culture is therefore necessary to enable progress. Additionally, while some new ideas from existing Joint doctrine have been acknowledged, this paper will examine additional options for Civil Support C2.

During the past several years, it has become clear that small RNAs guard germ cell genomes from the activity of mobile genetic elements. Indeed, in mammals, a class of small RNAs, known as Piwi-interacting RNAs (piRNAs), forms an innate immune system that discriminates transposons from endogenous genes and selectively silences the former. piRNAs enforce silencing by directing transposon DNA methylation during male germ cell development. As such, piRNAs represent perhaps the only currently known sequence-specific factor for deposition of methylcytosine in mammals. The three mammalian Piwi proteins Miwi2, Mili, and Miwi are required at different stages of germ cell development. Moreover, distinct classes of piRNAs are expressed in developmental waves, with particular generative loci and different sequence content distinguishing piRNAs populations in embryonic germ cells from those that appear during meiosis. Although our understanding of Piwi proteins and piRNA biology have deepened substantially during the last several years, major gaps still exist in our understanding of these enigmatic RNA species.

We study the process of DNA replication in proliferating human cells. Our efforts are directed to the identification and characterization of proteins that promote DNA replication ("initiators") as well as the DNA sequences recognized by them ("replicators") . We have focused in a group of initiator proteins called Origin Recognition Complex (ORC), Cdc6, and the Mini-Chromosome Maintenance (MCM) proteins. hOrclp, the largest subunit of ORC, appears to be a critical factor for the coordination of DNA replication with the cell division cycle. hOrclp levels are higher between the exit of mitosis and the end of Cl, the stage at which initiator proteins are assembled at the origins of replication. As cells enter S phase, hOrclp is polyubiquitinated on chromatin and degraded by the proteasome. hOrclp destruction is signaled in part by the SCFskP2 ubiquitin conjugating machinery. The controlled destruction of hOrclp likely contributes to avoid DNA overreplication, a common phenomenon in cancer cells. Our efforts to identify DNA replicator sequences using chromatin immunoprecipitation (ChIP) assays support the hypothesis that human origins of replication may not be defined by short specific DNA sequences but rather by higher-order chromatin structures.

Our goal is to understand the process of DNA replication at the molecular level in human cells. We are specifically trying to identify human replicator sequences and to characterize the pre-replicative complexes at the origins of replication. In this report we summarize a series of experiments using chromatin immunoprecipitation techniques and discuss the technical problems associated with them. Then we evaluate the stability of the human origin recognition complex across the cell cycle and present a novel regulation of its large subunit.

Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence. These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein, blocked the 5 phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G1-specific Cdc28p-Cln2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.

Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence. These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein, blocked the 5 phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G1-specific Cdc28p-Cln2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.

Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence. These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein, blocked the 5 phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G1-specific Cdc28p-Cln2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.

The coordination between cellular DNA replication and mitosis is critical to ensure controlled cell proliferation and accurate transmission of the genetic information as cells divide -two aspects of cellular life tipically lost in cancer. In order to unravel the molecular mechanisms of human DNA replication in normal and cancer cells, we have started a search for human DNA sequences that serve as replicators", this is, binding sites for human proteins involved in the initiation of DNA replication. Preliminary results are presented.

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin- associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP- binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin- associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP- binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin- associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP- binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

The progression of pluripotent stem cells to differentiated cell lineages requires major shifts in cell differentiation programs. In both mammals and higher plants, this process appears to be controlled by a dedicated set of transcription factors, many of which are kingdom specific. These divergent transcription factors appear to operate, however, together with a shared suite of factors that affect the chromatin state. It is of major importance to investigate whether such shared global control mechanisms indicate a common mechanistic basis for preservation of the stem cell state, initiation of differentiation programs, and coordination of cell state transitions.

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P–3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.