Correction: Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity.

[This corrects the article DOI: 10.1371/journal.pone.0273198.].

Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries.

Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determine how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identify enrichment of three features of CTCF binding at strong TAD boundaries, consisting of strongly bound and closely spaced CTCF binding peaks, with a further enrichment of DNA-binding motifs within these peaks. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we establish that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered, individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong insulation between TADs.

The Drosophila Fab-7 boundary modulates Abd-B gene activity by guiding an inversion of collinear chromatin organization and alternate promoter use.

Hox genes encode transcription factors that specify segmental identities along the anteroposterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, a feature known as collinearity. In Drosophila, the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive changes in histone modifications, chromatin architecture, and use of boundary elements and cis-regulatory regions. To dissect functional hierarchies, we compare chromatin organization in cell lines and larvae, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary for insulation between 3D domains carrying different histone modifications. Interestingly, we detect a non-canonical inversion of collinear chromatin dynamics at Abd-B, with the domain of active histone modifications progressively decreasing in size. This dynamic chromatin organization differentially activates the alternative promoters of the Abd-B gene, thereby expanding the possibilities for fine-tuning of transcriptional output.

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs.

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.

Developmental stability: a major role for cyclin G in drosophila melanogaster.

Morphological consistency in metazoans is remarkable given the pervasive occurrence of genetic variation, environmental effects, and developmental noise. Developmental stability, the ability to reduce developmental noise, is a fundamental property of multicellular organisms, yet its genetic bases remains elusive. Imperfect bilateral symmetry, or fluctuating asymmetry, is commonly used to estimate developmental stability. We observed that Drosophila melanogaster overexpressing Cyclin G (CycG) exhibit wing asymmetry clearly detectable by sight. Quantification of wing size and shape using geometric morphometrics reveals that this asymmetry is a genuine-but extreme-fluctuating asymmetry. Overexpression of CycG indeed leads to a 40-fold increase of wing fluctuating asymmetry, which is an unprecedented effect, for any organ and in any animal model, either in wild populations or mutants. This asymmetry effect is not restricted to wings, since femur length is affected as well. Inactivating CycG by RNAi also induces fluctuating asymmetry but to a lesser extent. Investigating the cellular bases of the phenotypic effects of CycG deregulation, we found that misregulation of cell size is predominant in asymmetric flies. In particular, the tight negative correlation between cell size and cell number observed in wild-type flies is impaired when CycG is upregulated. Our results highlight the role of CycG in the control of developmental stability in D. melanogaster. Furthermore, they show that wing developmental stability is normally ensured via compensatory processes between cell growth and cell proliferation. We discuss the possible role of CycG as a hub in a genetic network that controls developmental stability.

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.

Regulation of Abd-B expression by Cyclin G and Corto in the abdominal epithelium of Drosophila.

Polycomb-group (PcG) and trithorax-group (trxG) genes encode important regulators of homeotic genes, repressors and activators, respectively. They act through epigenetic mechanisms that maintain chromatin structure. The corto gene of Drosophila melanogaster encodes a co-factor of these regulators belonging to the Enhancer of Trithorax and Polycomb class. We have previously shown that Corto maintains the silencing of the homeotic gene Abdominal-B in the embryo and that it interacts with a cyclin, Cyclin G, suggesting that it could be a major actor in the connection between Polycomb/Trithorax function and the cell cycle. We show here that inactivation of Cyclin G by RNA interference leads to rotated genitalia and cuticle defects in the posterior abdomen of pupae and that corto genetically interacts with Cyclin G for generating these phenotypes. Examination of these pupae shows that development of the dorsal histoblast nests that will give rise to the adult epithelium is impaired in the posterior segments which identity is specified by Abdominal-B. Using a line that expresses LacZ in the Abdominal-B domain, we show that corto maintains Abdominal-B repression in the pupal epithelium whereas Cyclin G maintains its activation. These results prompt us to propose that the interaction between the Enhancer of Trithorax and Polycomb Corto and Cyclin G is involved in regulating the balance between cell proliferation and cell differentiation during abdominal epithelium development.

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.

Dimerization of MLL fusion proteins immortalizes hematopoietic cells.

MLL fusion proteins are leukemogenic, but their mechanism is unclear. Induced dimerization of a truncated MLL immortalizes bone marrow and imposes a reversible block on myeloid differentiation associated with upregulation of Hox a7, a9, and Meis1. Both dimerized MLL and exon-duplicated MLL are potent transcriptional activators, suggesting a link between dimerization and partial tandem duplication of DNA binding domains of MLL. Dimerized MLL binds with higher affinity than undimerized MLL to a CpG island within the Hox a9 locus. However, MLL-AF9 is not dimerized in vivo. The data support a model in which either MLL dimerization/exon duplication or fusion to a transcriptional activator results in Hox gene upregulation and ultimately transformation.

Identification and characterization of polyhomeotic PREs and TREs.

The polyhomeotic (ph) gene is a member of the Polycomb group of genes (Pc-G), which are required for the maintenance of the spatial expression pattern of homeotic genes. In contrast to homeotic genes, ph is ubiquitously expressed and it is quantitatively regulated. ph is negatively regulated by the Pc-G genes, except Psc, and positively regulated by the antagonist trithorax group of genes (trx-G), suggesting that Pc-G and trx-G response elements (PREs and TREs) exist at the ph locus. In this study, we have functionally characterized PREs and TREs at the ph locus that function in transgenic constructs. We have identified a strong PRE and TRE in the ph proximal unit as well as a weak one in the ph distal unit. The PRE/TRE of both ph units appear atypical compared with the well-defined homeotic maintenance elements because the minimal ph proximal response element activity requires at least 2 kb of sequence and does not work at long range. We have used chromatin immunoprecipitation experiments on cultured cells and embryos to show that Pc-G proteins are located in restricted regions, close to the ph promoters that overlap functionally defined PRE/TREs. Our data suggest that ph PRE/TREs are cis-acting DNA elements that modulate rather than silence Pc-G- and trx-G-mediated regulation, enlarging the role of these two groups of genes in transcriptional regulation.

The leucine zipper motif of the Drosophila AF10 homologue can inhibit PRE-mediated repression: implications for leukemogenic activity of human MLL-AF10 fusions.

In a screen for Drosophila genes that interfere with transcriptional repression mediated by the Polycomb group of genes, we identified a dominant mutation affecting the Alhambra (Alh) gene, the fly homologue of the human AF10 gene. AF10 has been identified as a fusion partner of both MLL and CALM in infant leukemias. Both fusion proteins retain the leucine zipper domain of AF10 but not its PHD domain. We show here that, while the full-length ALH protein has no activity on Polycomb group-responsive elements (PREs), overexpression of the isolated ALH leucine zipper domain activates several PREs. Within the ALH full-length protein, the PHD domain inhibits the PRE deregulation mediated by the leucine zipper domain. This deregulation is conserved in the human AF10 leucine zipper domain, which confers the same activity on an oncogenic MLL-AF10 fusion protein expressed in Drosophila melanogaster. These data reveal new properties for the leucine zipper domain and thus might provide new clues to understanding the mechanisms by which AF10 fusion proteins in which the PHD domain is lost might trigger leukemias in humans.