Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability.

In Drosophila, ubiquitous expression of a short Cyclin G isoform generates extreme developmental noise estimated by fluctuating asymmetry (FA), providing a model to tackle developmental stability. This transcriptional cyclin interacts with chromatin regulators of the Enhancer of Trithorax and Polycomb (ETP) and Polycomb families. This led us to investigate the importance of these interactions in developmental stability. Deregulation of Cyclin G highlights an organ intrinsic control of developmental noise, linked to the ETP-interacting domain, and enhanced by mutations in genes encoding members of the Polycomb Repressive complexes PRC1 and PR-DUB. Deep-sequencing of wing imaginal discs deregulating CycG reveals that high developmental noise correlates with up-regulation of genes involved in translation and down-regulation of genes involved in energy production. Most Cyclin G direct transcriptional targets are also direct targets of PRC1 and RNAPolII in the developing wing. Altogether, our results suggest that Cyclin G, PRC1 and PR-DUB cooperate for developmental stability.

Drosophila Cyclin G and epigenetic maintenance of gene expression during development.

BACKGROUND: Cyclins and cyclin-dependent kinases (CDKs) are essential for cell cycle regulation and are functionally associated with proteins involved in epigenetic maintenance of transcriptional patterns in various developmental or cellular contexts. Epigenetic maintenance of transcription patterns, notably of Hox genes, requires the conserved Polycomb-group (PcG), Trithorax-group (TrxG), and Enhancer of Trithorax and Polycomb (ETP) proteins, particularly well studied in Drosophila. These proteins form large multimeric complexes that bind chromatin and appose or recognize histone post-translational modifications. PcG genes act as repressors, counteracted by trxG genes that maintain gene activation, while ETPs interact with both, behaving alternatively as repressors or activators. Drosophila Cyclin G negatively regulates cell growth and cell cycle progression, binds and co-localizes with the ETP Corto on chromatin, and participates with Corto in Abdominal-B Hox gene regulation. Here, we address further implications of Cyclin G in epigenetic maintenance of gene expression. RESULTS: We show that Cyclin G physically interacts and extensively co-localizes on chromatin with the conserved ETP Additional sex combs (ASX), belonging to the repressive PR-DUB complex that participates in H2A deubiquitination and Hox gene silencing. Furthermore, Cyclin G mainly co-localizes with RNA polymerase II phosphorylated on serine 2 that is specific to productive transcription. CycG interacts with Asx, PcG, and trxG genes in Hox gene maintenance, and behaves as a PcG gene. These interactions correlate with modified ectopic Hox protein domains in imaginal discs, consistent with a role for Cyclin G in PcG-mediated Hox gene repression. CONCLUSIONS: We show here that Drosophila CycG is a Polycomb-group gene enhancer, acting in epigenetic maintenance of the Hox genes Sex combs reduced (Scr) and Ultrabithorax (Ubx). However, our data suggest that Cyclin G acts alternatively as a transcriptional activator or repressor depending on the developmental stage, the tissue or the target gene. Interestingly, since Cyclin G interacts with several CDKs, Cyclin G binding to the ETPs ASX or Corto suggests that their activity could depend on Cyclin G-mediated phosphorylation. We discuss whether Cyclin G fine-tunes transcription by controlling H2A ubiquitination and transcriptional elongation via interaction with the ASX subunit of PR-DUB.

The elongin complex antagonizes the chromatin factor Corto for vein versus intervein cell identity in Drosophila wings.

Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG) and Enhancers of Trithorax and Polycomb (ETP) families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor--MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. We show here that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C), increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. We propose that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid.

New partners in regulation of gene expression: the enhancer of Trithorax and Polycomb Corto interacts with methylated ribosomal protein l12 via its chromodomain.

Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA-seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.

Drosophila melanogaster Cyclin G coordinates cell growth and cell proliferation.

Mammalian Cyclins G1 and G2 are unconventional cyclins whose role in regulating the cell cycle is ambiguous. Cyclin G1 promotes G2/M cell cycle arrest in response to DNA damage whereas ectopic expression of CCNG2, that encodes Cyclin G2, induces G1/S cell cycle arrest. The only Drosophila Cyclin G was previously shown to be a transcriptional regulator that interacts with the chromatin factor Corto and controls expression of the homeotic gene Abdominal B. It is very close to mammalian Cyclin G1 and G2 except in its N-terminal region, that interacts with Corto, and that seems to have been acquired in dipterans. Ubiquitous misregulation of Cyclin G (CycG) using transgenic lines lengthens development and induces phenotypes suggesting growth or proliferation defects. Using tissue-specific misregulation of CycG and FACS, we show that overproduction of Cyclin G produces small cells whereas shortage produces large cells, suggesting that Cyclin G negatively regulates cell growth. Furthermore, overexpression of CycG lengthens the cell cycle, with a prominent effect on G1 and S phases. Genetic interactions with Cyclin E suggest that Cyclin G prevents G1 to S transition and delays S phase progression. Control of cell growth and cell cycle by Cyclin G might be achieved via interaction with a network of partners, notably the cyclin-dependent kinases CDK4 and CDK2.

Involvement of the MP1 scaffold protein in ERK signaling regulation during Drosophila wing development.

Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved transduction pathways involved in many cellular processes. Kinase modules are associated with scaffold proteins that regulate signaling by providing critical spatial and temporal specificities. Some of these scaffold proteins have been shown to be conserved, both in sequence and function. In mouse, the scaffold MP1 (MEK Partner 1) forms a signaling complex with MEK1 and ERK1. In this work, we focus on Drosophila MP1 (dMP1). We show that dMP1 is expressed ubiquitously during embryonic and larval development. By in vitro and in vivo experiments, we show that dMP1 is located in the cytoplasm and the nuclei, and that it interacts with MEK and ERK. Genetic studies with transgenic Drosophila lines allowing either dMP1 over-expression or dMP1 down-regulation by RNA interference highlight dMP1 function in the control of cell differentiation during development of the Drosophila wing.

Implication of the Drosophila beta-amyloid peptide binding-like protein AMX in Notch signaling during early neurogenesis.

Lateral inhibition provides a mechanism to regulate neuroblast specification during early neurogenesis in Drososphila melanogaster embryos. This mechanism is mediated by the highly conserved Notch pathway. Defective lateral inhibition results in CNS hyperplasia at the expense of ectoderm development, hence genes causing this defect are called neurogenic. D. melanogaster almondex (amx) is a maternal neurogenic gene, crucially required for embryonic lateral inhibition. Genetic interaction studies previously revealed amx as a positive Notch pathway partner in several processes, acting potentially upstream of Notch. We show here that embryonic overexpression of Notch intracellular domain partially rescues maternal lack of amx, suggesting a role for AMX at the level of Notch processing. Our molecular data reveal that amx is ubiquitously expressed and encodes a conserved putative transmembrane protein, composed of several domains that are differently required for amx function in the fly. Sequence comparisons identify AMX as a Drosophila Beta-amyloid peptide Binding Protein (BBP) family member, a BBP-like protein or dBLP. Based on these data, we discuss the potential molecular function of AMX in early neurogenesis.

How Drosophila change their combs: the Hox gene Sex combs reduced and sex comb variation among Sophophora species.

Identification of the events responsible for rapid morphological variation during evolution can help understand how developmental processes are changed by genetic modifications and thus produce diverse body features and shapes. Sex combs, a sexually dimorphic structure, show considerable variation in morphology and numbers among males from related species of Sophophora, a subgenus of Drosophila. To address which evolutionary changes in developmental processes underlie this diversity, we first analyzed the genetic network that controls morphogenesis of a single sex comb in the model D. melanogaster. We show that it depends on positive and negative regulatory inputs from proximo-distal identity specifying genes, including dachshund, bric à brac, and sex combs distal. All contribute to spatial regulation of the Hox gene Sex combs reduced (Scr), which is crucial for comb formation. We next analyzed the expression of these genes in sexually dimorphic species with different comb numbers. Only Scr shows considerable expression plasticity, which is correlated with comb number variation in these species. We suggest that differences in comb numbers reflect changes of Scr expression in tarsus primordia, and discuss how initial comb formation could have occurred in an ancestral Sophophora fly following regulatory modifications of developmental programs both parallel to and downstream of Scr.

Differential requirements for the neurogenic gene almondex during Drosophila melanogaster development.

During early development, the neurogenic genes of Drosophila melanogaster are involved in the control of cell fates in the neurectoderm; almondex (amx) belongs to this category of genes. We have identified the amx locus and rescued the amx embryonic neurogenic phenotype with a 1.5 kb DNA fragment. Using a small deficiency, we generated a new amx mutant background called amx(m), which is a null allele. Besides the characteristic neurogenic maternal effect caused by loss of amx, amx(m) flies display a new imaginal phenotype resembling loss of function of Notch. We describe amx-induced misregulation of the Notch pathway target E(spl) m7 in embryos and genetic interactions between amx and Notch pathway mutants in adult flies. These data show that wildtype amx acts as a novel positive regulator of the Notch pathway and is required at different levels during development.

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene.

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.

The multi sex combs gene of Drosophila melanogaster is required for proliferation of the germline.

We present a genetic analysis showing that the Drosophila melanogaster gene multi sex combs (mxc; Santamaria and Randsholt 1995) is needed for proliferation of the germline. Fertility is the feature most easily affected by weak hypomorphic mutations of this very pleiotropic locus. Pole cell formation and early steps of gonadogenesis conform to the wild-type in embryos devoid of zygotic mxc + product. mxc mutant gonad phenotypes and homozygous mxc germline clones suggest a role for mxc + in control of germ cell proliferation during the larval stages. mxc + requirement is germ cell autonomous and specific in females, whilst in males mxc + product is also needed in somatic cells of the gonads. Although mxc can be classified among the Polycomb group (Pc-G) of genes, negative trans-regulators of the ANT-C and BX-C gene complexes, germline requirement for mxc appears independent of a need for other Pc-C gene products, and mxc gonad phenotypes are different from those induced by mutations in BX-C genes. We discuss the possible functions of the mxc + product which helps to maintain homeotic genes repressed and prevents premature larval haemocyte differentiation and neoplasic overgrowth, but promotes growth and differentiation of male and female gonads.

Pattern triplications following genetic ablation on the wing ofDrosophila : Effect of eliminating thepolyhomeotic gene.

The effects ofpolyhomeotic (ph) mutants in imaginal cells have been studied in a clonal analysis. Clones of cells, homozygous forph, sort-out after a few divisions, probably as a consequence of modified cell affinities. The dorso-ventral margin of the wing has special characteristics that retard this phenomenon. The formation and exclusion of a clone of 8-16 cells affect the polarity of the wild-type neighbour cells and can provoke pattern triplications. The results suggest that a defect in intercellular communication prevents the wild-type cells from maintaining coordinated positional information. The cells react by regenerative growth, and reorganize into a new pattern. The pleiotropic phenotypes ofph mutants are explained according to a common hypothesis aboutph + function.

Maternal and zygotic requirement for thepolyhomeotic complex genetic locus inDrosophila.

The complex genetic locuspolyhomeotic (ph) is a member of thePolycomb (Pc)-group of genes and as such is required for the normal expression of ANT-C and BX-C genes. It also has probably other functions since amorphicph alleles display a cell death phenotype in the ventral epidermis of 12-h-old embryos. Here it is shown that lethal alleles ofph (amorph and strong hypomorph) show transformation of most of their segments towards AB8. Theph + product is required autonomously in imaginal cells. The total lack ofph + function prevents viability of the cuticular derivatives of these cells.ph has a strong maternal effect on segmental identity and epidermal development that can not be rescued by one paternally supplied dose ofph + in the zygote. These phenotypes differ substantially from those of previously describedPc-group genes. AmongPc-group genes,ph seems to be the only one that is strongly required both maternally and zygotically for normal embryonic development.