Sander van den Heuvel

GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes.

Publisher Summary Complete genome sequences are available for three model organisms— Escherichia coli , Saccharomyces cerevisiae , and Caenorhabditis elegans —and for several pathogenic microorganisms such as Helicobacter pylori . Complete genome sequences are expected to become available soon for other model organisms and for humans. This information is expected to revolutionize the way biological questions can be addressed. Molecular mechanisms should now be approachable on a more global scale in the context of (nearly) complete sets of genes, rather than by analyzing genes individually. However, most open reading frames (ORFs) predicted from sequencing projects have remained completely uncharacterized at the functional level. The emerging field of functional genomics addresses this limitation by developing methods to characterize the function of large numbers of predicted ORFs simultaneously.

Conserved functions of the pRB and E2F families.

Proteins that are related to the retinoblastoma tumour suppressor pRB and the E2F transcription factor are conserved in many species of plants and animals. The mammalian orthologues of pRB and E2F are best known for their roles in cell proliferation, but it has become clear that they affect many biological processes. Here we describe the functions of pRB-related proteins and E2F proteins that have emerged from genetic and biochemical experiments in Caenorhabditis elegans and Drosophila melanogaster. The similarities that have been observed between worms, flies and mammals provide insight into the core activities of pRB and E2F proteins and show how a common regulatory module can control various biological functions in different organisms.

An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis

The sterol regulatory element binding protein (SREBP) family of transcription activators are critical regulators of cholesterol and fatty acid homeostasis. We previously demonstrated that human SREBPs bind the CREB-binding protein (CBP)/p300 acetyltransferase KIX domain and recruit activator-recruited co-factor (ARC)/Mediator co-activator complexes through unknown mechanisms. Here we show that SREBPs use the evolutionarily conserved ARC105 (also called MED15) subunit to activate target genes. Structural analysis of the SREBP-binding domain in ARC105 by NMR revealed a three-helix bundle with marked similarity to the CBP/p300 KIX domain. In contrast to SREBPs, the CREB and c-Myb activators do not bind the ARC105 KIX domain, although they interact with the CBP KIX domain, revealing a surprising specificity among structurally related activator-binding domains. The Caenorhabditis elegans SREBP homologue SBP-1 promotes fatty acid homeostasis by regulating the expression of lipogenic enzymes. We found that, like SBP-1, the C. elegans ARC105 homologue MDT-15 is required for fatty acid homeostasis, and show that both SBP-1 and MDT-15 control transcription of genes governing desaturation of stearic acid to oleic acid. Notably, dietary addition of oleic acid significantly rescued various defects of nematodes targeted with RNA interference against sbp-1 and mdt-15, including impaired intestinal fat storage, infertility, decreased size and slow locomotion, suggesting that regulation of oleic acid levels represents a physiologically critical function of SBP-1 and MDT-15. Taken together, our findings demonstrate that ARC105 is a key effector of SREBP-dependent gene regulation and control of lipid homeostasis in metazoans.

DOCK4, a GTPase activator, is disrupted during tumorigenesis

We used representational difference analysis to identify homozygous genomic deletions selected during tumor progression in the mouse NF2 and TP53 tumor model. We describe a deletion targeting DOCK4, a member of the CDM gene family encoding regulators of small GTPases. DOCK4 specifically activates Rap GTPase, enhancing the formation of adherens junctions. DOCK4 mutations are present in a subset of human cancer cell lines; a recurrent missense mutant identified in human prostate and ovarian cancers encodes a protein that is defective in Rap1 activation. The engulfment defect of C. elegans mutants lacking the CDM gene ced-5 is rescued by wild-type DOCK4, but not by the mutant allele. Expression of wild-type, but not mutant, DOCK4 in mouse osteosarcoma cells with a deletion of the endogenous gene suppresses growth in soft agar and tumor invasion in vivo. DOCK4 therefore encodes a CDM family member that regulates intercellular junctions and is disrupted during tumorigenesis.

CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.

S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells

ABSTRACT Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies inXenopus egg extracts and microinjection studies in human cells have suggested an additional function for Cdk2 in activation of Cdk1 and entry into mitosis. To further examine the role of Cdk2 in human cells, we generated stable clones with inducible expression of wild-type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Both exogenous proteins associated efficiently with endogenous cyclins. Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn inhibited progression through several distinct stages. Cdk2-dn induction could arrest cells at the G1/S transition, as previously observed in transient expression studies. However, under normal culture conditions, Cdk2-dn induction primarily arrested cells with S and G2/M DNA contents. Several observations suggested that the latter cells were in G2 phase, prior to the onset of mitosis: these cells contained uncondensed chromosomes, low levels of cyclin B-associated kinase activity, and high levels of tyrosine-phosphorylated Cdk1. Furthermore, Cdk2-dn did not delay progression through mitosis upon release of cells from a nocodazole block. Although the G2arrest imposed by Cdk2-dn was similar to that imposed by the DNA damage checkpoint, the former was distinguished by its resistance to caffeine. These findings provide evidence for essential functions of Cdk2 during S and G2 phases of the mammalian cell cycle.

C. elegans Class B Synthetic Multivulva Genes Act in G1 Regulation

The single C. elegans member of the retinoblastoma gene family, lin-35 Rb, was originally identified as a synthetic Multivulva (synMuv) gene [1]. These genes form two redundant classes, A and B, that repress ectopic vulval cell fate induction. Recently, we demonstrated that lin-35 Rb also acts as a negative regulator of G(1) progression and likely is the major target of cyd-1 Cyclin D and cdk-4 CDK4/6. Here, we describe G(1) control functions for several other class B synMuv genes. We found that efl-1 E2F negatively regulates cell cycle entry, while dpl-1 DP appeared to act both as a positive and negative regulator. In addition, we identified a negative G(1) regulatory function for lin-9 ALY, as well as lin-15B and lin-36, which encode novel proteins. Inactivation of lin-35 Rb, efl-1, or lin-36 allowed S phase entry in the absence of cyd-1/cdk-4 and increased ectopic cell division when combined with cki-1 Cip/Kip RNAi. These data are consistent with lin-35 Rb, efl-1, and lin-36 acting in a common pathway or complex that negatively regulates G(1) progression. In contrast, lin-15B appeared to act in parallel to lin-35. Our results demonstrate the potential for genetic identification of novel G(1) regulators in C. elegans.

The Conserved Kinases CDK-1, GSK-3, KIN-19, and MBK-2 Promote OMA-1 Destruction to Regulate the Oocyte-to-Embryo Transition in C. elegans

BACKGROUND At the onset of embryogenesis, key developmental regulators called determinants are activated asymmetrically to specify the body axes and tissue layers. In C. elegans, this process is regulated in part by a conserved family of CCCH-type zinc finger proteins that specify the fates of early embryonic cells. The asymmetric localization of these and other determinants is regulated in early embryos through motor-dependent physical translocation as well as selective proteolysis. RESULTS We show here that the CCCH-type zinc finger protein OMA-1 serves as a nexus for signals that regulate the transition from oogenesis to embryogenesis. While OMA-1 promotes oocyte maturation during meiosis, destruction of OMA-1 is needed during the first cell division for the initiation of ZIF-1-dependent proteolysis of cell-fate determinants. Mutations in four conserved protein kinase genes-mbk-2/Dyrk, kin-19/CK1alpha, gsk-3, and cdk-1/CDC2-cause stabilization of OMA-1 protein, and their phenotypes are partially suppressed by an oma-1 loss-of-function mutation. OMA-1 proteolysis also depends on Cyclin B3 and on a ZIF-1-independent CUL-2-based E3 ubiquitin ligase complex, as well as the CUL-2-interacting protein ZYG-11 and the Skp1-related proteins SKR-1 and SKR-2. CONCLUSIONS Our findings suggest that a CDK1/Cyclin B3-dependent activity links OMA-1 proteolysis to completion of the first cell cycle and support a model in which OMA-1 functions to prevent the premature activation of cell-fate determinants until after they are asymmetrically partitioned during the first mitosis.

NuMA-related LIN-5, ASPM-1, calmodulin and dynein promote meiotic spindle rotation independently of cortical LIN-5/GPR/Galpha

The spindle apparatus dictates the plane of cell cleavage, which is critical in the choice between symmetric or asymmetric division. Spindle positioning is controlled by an evolutionarily conserved pathway, which involves LIN-5/GPR-1/2/Gα in Caenorhabditis elegans, Mud/Pins/Gα in Drosophila and NuMA/LGN/Gα in humans. GPR-1/2 and Gα localize LIN-5 to the cell cortex, which engages dynein and controls the cleavage plane during early mitotic divisions in C. elegans. Here we identify ASPM-1 (abnormal spindle-like, microcephaly-associated) as a novel LIN-5 binding partner. ASPM-1, together with calmodulin (CMD-1), promotes meiotic spindle organization and the accumulation of LIN-5 at meiotic and mitotic spindle poles. Spindle rotation during maternal meiosis is independent of GPR-1/2 and Gα, yet requires LIN-5, ASPM-1, CMD-1 and dynein. Our data support the existence of two distinct LIN-5 complexes that determine localized dynein function: LIN-5/GPR-1/2/Gα at the cortex, and LIN-5/ASPM-1/CMD-1 at spindle poles. These functional interactions may be conserved in mammals, with implications for primary microcephaly.

SUMO modification is required for in vivo Hox gene regulation by the Caenorhabditis elegans Polycomb group protein SOP-2

Post-translational modification of proteins by the ubiquitin-like molecule SUMO (sumoylation) regulates their subcellular localization and affects their functional properties in vitro, but the physiological function of sumoylation in multicellular organisms is largely unknown. Here, we show that the C. elegans Polycomb group (PcG) protein SOP-2 interacts with the SUMO-conjugating enzyme UBC-9 through its evolutionarily conserved SAM domain. Sumoylation of SOP-2 is required for its localization to nuclear bodies in vivo and for its physiological repression of Hox genes. Global disruption of sumoylation phenocopies a sop-2 mutation by causing ectopic Hox gene expression and homeotic transformations. Chimeric constructs in which the SOP-2 SAM domain is replaced with that derived from fruit fly or mammalian PcG proteins, but not those in which the SOP-2 SAM domain is replaced with the SAM domains of non-PcG proteins, confer appropriate in vivo nuclear localization and Hox gene repression. These observations indicate that sumoylation of PcG proteins, modulated by their evolutionarily conserved SAM domain, is essential to their physiological repression of Hox genes.