Joachim Marhold

Kremen proteins are Dickkopf receptors that regulate Wnt/|[beta]|-catenin signalling

The Wnt family of secreted glycoproteins mediate cell–cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the β-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular β-catenin. Wnt/β-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/β-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.

Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation

DNA methylation is an epigenetic modification that is essential for gene silencing and genome stability in many organisms. Although methyltransferases that promote DNA methylation are well characterized, the molecular mechanism underlying active DNA demethylation is poorly understood and controversial. Here we show that Gadd45a (growth arrest and DNA-damage-inducible protein 45 alpha), a nuclear protein involved in maintenance of genomic stability, DNA repair and suppression of cell growth, has a key role in active DNA demethylation. Gadd45a overexpression activates methylation-silenced reporter plasmids and promotes global DNA demethylation. Gadd45a knockdown silences gene expression and leads to DNA hypermethylation. During active demethylation of oct4 in Xenopus laevis oocytes, Gadd45a is specifically recruited to the site of demethylation. Active demethylation occurs by DNA repair and Gadd45a interacts with and requires the DNA repair endonuclease XPG. We conclude that Gadd45a relieves epigenetic gene silencing by promoting DNA repair, which erases methylation marks.

A Dnmt2-like protein mediates DNA methylation in Drosophila.

The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation, we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins.

The DrosDel Deletion Collection: A Drosophila Genomewide Chromosomal Deficiency Resource

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering ∼77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.

The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA

Methyl-DNA binding proteins play an important role in epigenetic gene regulation. The Drosophila genome encodes a single protein (MBD2/3) with extended homologies to the vertebrate methyl-DNA binding proteins MBD2 and MBD3. However, very little is known about its functional properties. We have now characterized an MBD2/3 null mutant allele that is viable and fertile. This mutation caused a strong dominant suppression of position-effect variegation and also resulted in a high rate of chromosome segregation defects during early embryogenesis. Confocal analysis of mutant embryos showed local displacement of MI-2 from DNA and indicated that MBD2/3 is associated with only a subset of MI-2 complexes. In addition, band shift experiments demonstrated a specific binding of MBD2/3 to CpT/A-methylated DNA, which reflects the endogenous DNA methylation pattern of Drosophila. Consistently, the localization of MBD2/3 was disrupted in embryos with reduced levels of DNA methylation. Our data provide novel insights into the function of MBD2/3 proteins and strongly suggest the existence of methylation-dependent chromatin structures in Drosophila.

The Drosophila methyl-DNA binding protein MBD2/3 interacts with the NuRD complex via p55 and MI-2

BackgroundMethyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications. MBD2/3 is the only candidate gene in the Drosophila genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the N ucleosome R emodelling and D eacetylase (NuRD) complex, respectively. An association of Drosophila MBD2/3 with the Drosophila NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail.ResultsThe two MBD2/3 isoforms precisely cofractionated with NuRD proteins during gel filtration of extracts derived from early and late embryos. In addition, we demonstrate that MBD2/3 forms multimers, and engages in specific interactions with the p55 and MI-2 subunits of the Drosophila NuRD complex.ConclusionOur data provide novel insights into the association between Drosophila MBD2/3 and NuRD proteins. Additionally, this work provides a first analysis of the architecture of the Drosophila NuRD complex.

Stage-specific chromosomal association of Drosophila dMBD2/3 during genome activation

Abstract. The Drosophila gene dMBD2/3 encodes a protein with significant homologies to the mammalian methyl-DNA binding proteins MBD2 and MBD3. These proteins are essential components of chromatin complexes involved in epigenetic gene regulation. Because the available in vitro data on dMBD2/3 are conflicting we have started an in vivo characterization of dMBD2/3. We detected expression of two isoforms specifically during embryonic development. Staining of whole embryos combined with high-resolution confocal microscopy revealed a highly regulated spatial distribution. During the syncytial blastoderm stage, dMBD2/3 formed speckles that localized to the cytoplasm. Shortly after, during the cellular blastoderm stage, the protein entered the nucleus and formed bright foci that associated with DNA. This rapid transition coincided with the activation of the embryonic genome. A similar observation was made during activation of the spermatocyte genome as dMBD2/3 formed distinct foci associated with the activated Y chromosome. Our results indicate that dMBD2/3 forms specialized nuclear compartments to keep certain genes epigenetically silenced during genome activation.

Drosophila spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries

Background: Mammalian RNA polymerase II-associated protein 3 (RPAP3) recruits heat shock protein 90 (Hsp90) to assemble cellular machineries such as RNA polymerases. Results: Spaghetti encodes the Drosophila homolog of RPAP3. Spaghetti is essential for development. Spag protein binds and stimulates Hsp90 and Hsp70. Conclusion: RPAP3 function is conserved among metazoans. Significance: Our data suggest that Hsp70 assists RPAP3 in complex assembly. The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA+-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.

Differential expression of two scribble isoforms during Drosophila embryogenesis.

The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones.

Requirements for scribble expression in newly formed gonads of Drosophila embryos.

The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila, and encodes two protein isoforms. Here, we report the pattern of Scrib1 synthesis in pole cells and embryonic gonads. We found that Scrib1 synthesis became strongly enhanced in pole cells at the time of gonad formation and was also detectable in cortical domains of gonadal mesodermal cells adjacent to pole cells. Scrib1 synthesis in mesodermal cells was independent of pole cells and occurred in agametic valois and capsuléen embryonic gonads. In contrast, Scrib1 synthesis in pole cells required contact with gonadal mesodermal cells as revealed by the absence of Scrib1 in wunen or tinman-zinc finger homeodomain-1 pseudo-gonads made only of aggregated pole cells.