Anne Segonds-Pichon

Dynamic CpG island methylation landscape in oocytes and preimplantation embryos

Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming. Here we present, to our knowledge, the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L but are not distinct at the sequence level, including in CpG periodicity. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilization reprogramming but, notably, the majority show incomplete demethylation in embryonic day (E) 3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.

PI3Kβ Plays a Critical Role in Neutrophil Activation by Immune Complexes

The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases. The Integrating Isoform The class I phosphoinositide 3-kinases (PI3Ks) are implicated in processes such as growth factor signaling and inflammation. PI3Kγ is activated by G protein–coupled receptors (GPCRs), whereas PI3Kα and PI3Kδ are activated by protein tyrosine kinase–coupled receptors. PI3Kβ is unusual in that it appears to respond to signals from both types of receptors, depending on the cellular context. Kulkarni et al. investigated the responses of mouse neutrophils to immune complexes of antibody and antigen, which trigger chronic inflammation in conditions such as autoimmune arthritis. Genetic and pharmacological evidence suggested that immune complexes stimulated PI3Kβ in a process involving activation of FcγR, a tyrosine kinase–coupled low-affinity antibody receptor, and autocrine signaling by a proinflammatory lipid (LTB4) through its GPCR. Mice deficient in PI3Kβ fared better than did controls in models of arthritis and inflammatory skin disease. These data implicate PI3Kβ in the integration of signals from tyrosine kinase–coupled receptors and GPCRs—and suggest that this isoform may be an effective therapeutic target in inflammatory diseases. Neutrophils are activated by immunoglobulin G (IgG)–containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength–dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase–coupled receptor (FcγR) and a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies

Significance In eusocial insect societies, such as ants and some bees and wasps, phenotypes are highly plastic, generating alternative phenotypes (queens and workers) from the same genome. The greatest plasticity is found in simple insect societies, in which individuals can switch between phenotypes as adults. The genomic, transcriptional, and epigenetic underpinnings of such plasticity are largely unknown. In contrast to the complex societies of the honeybee, we find that simple insect societies lack distinct transcriptional differentiation between phenotypes and coherently patterned DNA methylomes. Instead, alternative phenotypes are largely defined by subtle transcriptional network organization. These traits may facilitate genomic plasticity. These insights and resources will stimulate new approaches and hypotheses that will help to unravel the genomic processes that create phenotypic plasticity. Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.

p110γ and p110δ isoforms of phosphoinositide 3-kinase differentially regulate natural killer cell migration in health and disease

The mechanisms that regulate NK cell trafficking are unclear. Phosphoinositide-3 kinases (PI3K) control cell motility and the p110γ and p110δ isoforms are mostly expressed in leukocytes, where they transduce signals downstream of G protein coupled receptors (GPCR) or tyrosine kinase receptors, respectively. Here, we set out to determine the relative contribution of p110γ and p110δ to NK cell migration in mice. Using a combination of single-cell imaging analysis of transgenic cells reporting on PI3K activity in real time and small molecule inhibitors of p110γ and p110δ, we show here that the tyrosine-kinase coupled p110δ is linked to GPCR signaling and, depending on the GPCR, may even be preferentially activated over p110γ. Using gene-targeted mice, we showed that both isoforms were essential for NK cell chemotaxis to CXCL12 and to CCL3 and, in vivo, for normal NK cell migration during pregnancy and to the inflamed peritoneum. By contrast, only p110δ was indispensable for chemotaxis to S1P and CXCL10 and for NK cell distribution throughout lymphoid and nonlymphoid tissues and for extravasation to tumors. These results implicate p110δ downstream of GPCRs in NK cells and highlight its nonredundant role as a key regulator of NK cell trafficking in health and disease.

P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination

The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2−/− and P-Rex1−/−/P-Rex2−/− mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2−/− pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2−/− and P-Rex1−/−/P-Rex2−/− mice. P-Rex2−/− mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1−/−/P-Rex2−/− mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function.

The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues

Summary The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.

The GTPase-activating protein ARAP3 regulates chemotaxis and adhesion-dependent processes in neutrophils

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased β2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating β2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated.

DNA methylation at differentially methylated regions of imprinted genes is resistant to developmental programming by maternal nutrition

The nutritional environment in which the mammalian fetus or infant develop is recognized as influencing the risk of chronic diseases, such as type 2 diabetes and hypertension, in a phenomenon that has become known as developmental programming. The late onset of such diseases in response to earlier transient experiences has led to the suggestion that developmental programming may have an epigenetic component, because epigenetic marks such as DNA methylation or histone tail modifications could provide a persistent memory of earlier nutritional states. One class of genes that has been considered a potential target or mediator of programming events is imprinted genes, because these genes critically depend upon epigenetic modifications for correct expression and because many imprinted genes have roles in controlling fetal growth as well as neonatal and adult metabolism. In this study, we have used an established model of developmental programming—isocaloric protein restriction to female mice during gestation or lactation—to examine whether there are effects on expression and DNA methylation of imprinted genes in the offspring. We find that although expression of some imprinted genes in liver of offspring is robustly and sustainably changed, methylation of the differentially methylated regions (DMRs) that control their monoallelic expression remains largely unaltered. We conclude that deregulation of imprinting through a general effect on DMR methylation is unlikely to be a common factor in developmental programming.

Social parasitism and the molecular basis of phenotypic evolution

Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in closely related species offer an unrivaled opportunity to evaluate the extent to which genomic material is reorganized to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled entities to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a eusocial ancestor and are specialized to exploit the socially acquired resources of their closely-related eusocial host. Molecular comparisons of such species pairs can reveal how genomic material is re-organized in the loss of ancestral traits (i.e., of free-living traits in the parasites) and the gain of new ones (i.e., specialist traits required for a parasitic lifestyle). We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in our understanding of the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data using the paper wasp social parasite/host system (Polistes sulcifer—Polistes dominula). This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic specialization.