Assessing the impact of transgenerational epigenetic variation on complex traits.

Loss or gain of DNA methylation can affect gene expression and is sometimes transmitted across generations. Such epigenetic alterations are thus a possible source of heritable phenotypic variation in the absence of DNA sequence change. However, attempts to assess the prevalence of stable epigenetic variation in natural and experimental populations and to quantify its impact on complex traits have been hampered by the confounding effects of DNA sequence polymorphisms. To overcome this problem as much as possible, two parents with little DNA sequence differences, but contrasting DNA methylation profiles, were used to derive a panel of epigenetic Recombinant Inbred Lines (epiRILs) in the reference plant Arabidopsis thaliana. The epiRILs showed variation and high heritability for flowering time and plant height ( approximately 30%), as well as stable inheritance of multiple parental DNA methylation variants (epialleles) over at least eight generations. These findings provide a first rationale to identify epiallelic variants that contribute to heritable variation in complex traits using linkage or association studies. More generally, the demonstration that numerous epialleles across the genome can be stable over many generations in the absence of selection or extensive DNA sequence variation highlights the need to integrate epigenetic information into population genetics studies.

A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae.

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.

The steroid-hormone ecdysone coordinates parallel pupariation neuromotor and morphogenetic subprograms via epidermis-to-neuron Dilp8-Lgr3 signal induction.

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.

CAF-1 is required for efficient replication of euchromatic DNA in Drosophila larval endocycling cells.

The endocycle constitutes an effective strategy for cell growth during development. In contrast to the mitotic cycle, it consists of multiple S-phases with no intervening mitosis and lacks a checkpoint ensuring the replication of the entire genome. Here, we report an essential requirement of chromatin assembly factor-1 (CAF-1) for Drosophila larval endocycles. This complex promotes histone H3-H4 deposition onto newly synthesised DNA in vitro. In metazoans, the depletion of its large subunit leads to the rapid accumulation of cells in S-phase. However, whether this slower S-phase progression results from the activation of cell cycle checkpoints or whether it reflects a more direct requirement of CAF-1 for efficient replication in vivo is still debated. Here, we show that, strikingly, Drosophila larval endocycling cells depleted for the CAF-1 large subunit exhibit normal dynamics of progression through endocycles, although accumulating defects, such as perturbation of nucleosomal organisation, reduction of the replication efficiency of euchromatic DNA and accumulation of DNA damage. Given that the endocycle lacks a checkpoint ensuring the replication of the entire genome, the biological context of Drosophila larval development offered a unique opportunity to highlight the requirement of CAF-1 for chromatin organisation and efficient replication processes in vivo, independently of checkpoint activation.

Deletion of H-Ras decreases renal fibrosis and myofibroblast activation following ureteral obstruction in mice.

Tubulointerstitial fibrosis is characterized by the presence of myofibroblasts that contribute to extracellular matrix accumulation. These cells may originate from resident fibroblasts, bone-marrow-derived cells, or renal epithelial cells converting to a mesenchymal phenotype. Ras GTPases are activated during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-beta-induced epithelial-mesenchymal transition. Here we set out to assess the contribution of Ras to experimental renal fibrosis using the well-established model of unilateral ureteral obstruction. Fifteen days after obstruction, both fibroblast proliferation and inducers of epithelial-mesenchymal transition were lower in obstructed kidneys of H-ras knockout mice and in fibroblast cell lines derived from these mice. Interestingly, fibronectin, collagen I accumulation, overall interstitial fibrosis, and the myofibroblast population were also lower in the knockout than in the wild-type mice. As expected, we found lower levels of activated Akt in the kidneys and cultured fibroblasts of the knockout. Whether Ras inhibition will turn out to prevent progression of renal fibrosis will require more direct studies.

Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing.

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.

A role for RNAi in the selective correction of DNA methylation defects.

DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, which suggests that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis (as in cancer) or meiosis, leading to epiallelic variation. We demonstrated the existence of an efficient mechanism that protects against transgenerational loss of DNA methylation in Arabidopsis. Remethylation is specific to the subset of heavily methylated repeats that are targeted by the RNA interference (RNAi) machinery. This process does not spread into flanking regions, is usually progressive over several generations, and faithfully restores wild-type methylation over target sequences in an RNAi-dependent manner. Our findings suggest an important role for RNAi in protecting genomes against long-term epigenetic defects.

First evidence of methylation in the genome of Drosophila willistoni.

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogaster dDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.

Complex evolution of gypsy in Drosophilid species.

In an endeavor to contribute to the comprehension of the evolution of transposable elements (TEs) in the genome of host species, we investigated the phylogenetic relationships of sequences homologous to the retrotransposon gypsy of Drosophila melanogaster in 19 species of Drosophila, in Scaptodrosophila latifasciaeformis, and in Zaprionus indianus. This phylogenetic study was based on approximately 500 base pairs of the env gene. Our analyses showed considerable discrepancy between the phylogeny of gypsy elements and the relationship of their host species, and they allow us to infer a complex evolutionary pattern that could include ancestral polymorphism, vertical transmission, and several cases of horizontal transmission.

Essentiality and damage in metabolic networks.

Understanding the architecture of physiological functions from annotated genome sequences is a major task for postgenomic biology. From the annotated genome sequence of the microbe Escherichia coli, we propose a general quantitative definition of enzyme importance in a metabolic network. Using a graph analysis of its metabolism, we relate the extent of the topological damage generated in the metabolic network by the deletion of an enzyme to the experimentally determined viability of the organism in the absence of that enzyme. We show that the network is robust and that the extent of the damage relates to enzyme importance. We predict that a large fraction (91%) of enzymes causes little damage when removed, while a small group (9%) can cause serious damage. Experimental results confirm that this group contains the majority of essential enzymes. The results may reveal a universal property of metabolic networks.

Distribution and conservation of the transposable element gypsy in drosophilid species

In an attempt to understand the dynamics of transposable elements (T'S) in the genome of host species, we investigated the distribution, representativeness and conservation of DNA sequences homologous to the Drosophila melanogaster gypsy retrotransposon in 42 drosophilid species. Our results extended the knowledge about the wide distribution of gypsy in the genus Drosophila, including several Neotropical species not previously studied. The gypsy-like sequences showed high divergence compared to the D. melanogaster gypsy element. Furthermore, the conservation of the restriction sites between gypsy sequences from phylogenetically unrelated species pointed to a more complex evolutionary picture, which includes the possibility of the horizontal transfer events already described for this retrotransposon.

Transposable elements P and gypsy in natural populations of Drosophila willistoni

The presence and integrity of the P transposon and the gypsy retrotransposon in the genome of 18 samples of natural Drosophila willistoni populations collected from a large area of South America were Southern blot screened using Drosophila melanogaster probes. The aim of this screening was provide further knowledge-base on the geographical distribution of D. willistoni and to carry out an inter-population analysis of the P and gypsy elements present in the genomes of the populations analyzed. The fragment patterns obtained indicate that both the P and gypsy elements are ancient in the D. willistoni genome, but whereas the gypsy retroelement appears to be invariable and stable the P element varies between populations and appears to still have some capacity for mobilization.