The enhancer of trithorax and polycomb corto interacts with cyclin G in Drosophila

Background

Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called “Enhancers of Trithorax and Polycomb” (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown.

Methodology/Principal Findings

In a two-hybrid screen, we identified Cyclin G (CycG) as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. We show here that Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). We observed that corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene.

Conclusions/Significance

Our results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG.     

The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development

Background  

Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here.     

Rm62, a DEAD‐box RNA helicase,complexes with DSP1 in Drosophila embryos

Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB‐like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD‐box RNA helicase, Rm62, as partner of DSP1 in a 250‐kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3‐ to 12‐h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes. genesis 48:244–253, 2010. © 2010 Wiley‐Liss, Inc.     

The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish 'bivalent chromatin marks' at the silent AGAMOUS locus

Tightly balanced antagonism between the Polycomb group (PcG) and the Trithorax group (TrxG) complexes maintain Hox expression patterns in Drosophila and murine model systems. Factors belonging to the PcG/TrxG complexes control various processes in plants as well but whether they participate in mechanisms that antagonize, balance or maintain each other's effects at a particular gene locus is unknown. CURLY LEAF (CLF), an Arabidopsis homolog of enhancer of zeste (EZ) and the ARABIDOPSIS HOMOLOG OF TRITHORAX (ATX1) control the expression of the flower homeotic gene AGAMOUS (AG). Disrupted ATX1 or CLF function results in misexpression of AG, recognizable phenotypes and loss of H3K4me3 or H3K27me3 histone H3-tail marks, respectively. A novel idea suggested by our results here, is that PcG and TrxG complexes function as a specific pair generating bivalent chromatin marks at the silent AG locus. Simultaneous loss of ATX1 and CLF restored AG repression and normalized leaf phenotypes. At the molecular level, disrupted ATX1 and CLF functions did not lead to erasure of the CLF- and ATX1-generated epigenetic marks, as expected: instead, in the double mutants, H3K27me3 and H3K4me3 tags were partially restored. We demonstrate that ATX1 and CLF physically interact linking mechanistically the observed effects.     

Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin

The Polycomb (PcG) and Trithorax (TrxG) group proteins work antagonistically on several hundred developmentally important target genes, giving stable mitotic memory, but also allowing flexibility of gene expression states. How this is achieved in quantitative terms is poorly understood. Here, we present a quantitative kinetic analysis in living Drosophila of the PcG proteins Enhancer of Zeste, (E(Z)), Pleiohomeotic (PHO) and Polycomb (PC) and the TrxG protein absent, small or homeotic discs 1 (ASH1). Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy reveal highly dynamic chromatin binding behaviour for all proteins, with exchange occurring within seconds. We show that although the PcG proteins substantially dissociate from mitotic chromatin, ASH1 remains robustly associated with chromatin throughout mitosis. Finally, we show that chromatin binding by ASH1 and PC switches from an antagonistic relationship in interphase, to a cooperative one during mitosis. These results provide quantitative insights into PcG and TrxG chromatin-binding dynamics and have implications for our understanding of the molecular nature of epigenetic memory.     

The detached locus encodes Drosophila Dystrophin, which acts with other components of the Dystrophin Associated Protein Complex to influence intercellular signalling in developing wing veins

Dystrophin and Dystroglycan are the two central components of the multimeric Dystrophin Associated Protein Complex, or DAPC, that is thought to provide a mechanical link between the extracellular matrix and the actin cytoskeleton, disruption of which leads to muscular dystrophy in humans. We present the characterization of the Drosophila ‘crossveinless’ mutation detached (det), and show that the gene encodes the fly ortholog of Dystrophin. Our genetic analysis shows that, in flies, Dystrophin is a non-essential gene, and the sole overt morphological defect associated with null mutations in the locus is the variable loss of the posterior crossvein that has been described for alleles of det. Null mutations in Drosophila Dystroglycan (Dg) are similarly viable and exhibit this crossvein defect, indicating that both of the central DAPC components have been co-opted for this atypical function of the complex. In the developing wing, the Drosophila DAPC affects the intercellular signalling pathways involved in vein specification. In det and Dg mutant wings, the early BMP signalling that initiates crossvein specification is not maintained, particularly in the pro-vein territories adjacent to the longitudinal veins, and this results in the production of a crossvein fragment in the intervein between the two longitudinal veins. Genetic interaction studies suggest that the DAPC may exert this effect indirectly by down-regulating Notch signalling in pro-vein territories, leading to enhanced BMP signalling in the intervein by diffusion of BMP ligands from the longitudinal veins.     

Genetic and epigenetic mechanisms regulating hedgehog expression in the Drosophila wing

Stable subdivision of Drosophila limbs into Anterior (A) and Posterior (P) compartments is a consequence of asymmetric signaling by Hedgehog (Hh) from P to A cells. The activity of the homeodomain protein Engrailed (En) in P cells has been reported to help to generate this asymmetry by inducing the expression of hedgehog and simultaneously repressing the expression of the essential downstream component of the Hh signaling pathway Cubitus interruptus (Ci). In A cells, Ci has a major role in the repression of hh. Here we have revised the genetic and epigenetic mechanisms involved in the regulation of hh in the P compartment. First, we present evidence that hh expression in P cells is a consequence of the repression of ci by the activity of En. Thus, in the absence of Ci and En activities, cells do express hh. We also present data supporting the maintenance of hh expression in P cells through epigenetic mechanisms, and a permissive role of Notch signaling in this process. Notch and Trithorax (TrxG) group of proteins exert their action through a previously defined hh Polycomb Responsive Element (PRE).     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.