Impact of food restriction on the medio-basal hypothalamus of intact ewes as revealed by a large-scale transcriptomics study.

In mammals, the medio-basal hypothalamus (MBH) integrates photoperiodic and food-related cues to ensure timely phasing of physiological functions, including seasonal reproduction. The current human epidemics of obesity and associated reproductive disorders exemplifies the tight link between metabolism and reproduction. Yet, how food-related cues impact breeding at the level of the MBH remains unclear. In this respect, the sheep, which is a large diurnal mammal with a marked dual photoperiodic/metabolic control of seasonal breeding, is a relevant model. Here, we present a large-scale study in ewes (n = 120), which investigated the impact of food restriction (FRes) on the MBH transcriptome using unbiased RNAseq, followed by RT-qPCR. Few genes (~100) were impacted by FRes and the transcriptional impact was very modest (<2-fold increase or < 50% decrease for most genes). As anticipated, FRes increased expression of Npy/AgRP/LepR and decreased expression of Pomc/Cartpt, while Kiss1 expression was not impacted. Of particular interest, Eya3, Nmu and Dio2, genes involved in photoperiodic decoding within the MBH, were also affected by FRes. Finally, we also identified a handful of genes not known to be regulated by food-related cues (e.g., RNase6, HspA6, Arrdc2). In conclusion, our transcriptomics study provides insights into the impact of metabolism on the MBH in sheep, which may be relevant to human, and identifies possible molecular links between metabolism and (seasonal) reproduction.

Mesorhizobium ventifaucium sp. nov. and Mesorhizobium escarrei sp. nov., two novel root-nodulating species isolated from Anthyllis vulneraria.

Ten mesorhizobial strains isolated from root-nodules of Anthyllis vulneraria by trapping using soils from southern France were studied to resolve their taxonomy. Their 16S rDNA sequences were identical and indicated that they are affiliated to the genus Mesorhizobium within the group M. prunaredense/M. delmotii/M. temperatum/M. mediterraneum/M. wenxiniae and M. robiniae as the closest defined species. Their evolutionary relationships with validated species were further characterized by multilocus sequence analysis (MLSA) using 4 protein-coding housekeeping genes (recA, atpD, glnII and dnaK), that divides the strains in two groups, and suggest that they belong to two distinct species. These results were well-supported by MALDI-TOF mass spectrometry analyses, wet-lab DNA-DNA hybridization (≤58%), and genome-based species delineation methods (ANI < 96%, in silico DDH < 70%), confirming their affiliation to two novel species. Based on these differences, Mesorhizobium ventifaucium (STM4922T = LMG 29643T = CFBP 8438T) and Mesorhizobium escarrei (type strain STM5069T = LMG 29642T = CFBP 8439T) are proposed as names for these two novel species. The phylogeny of nodulation genes nodC and nodA allocated the type strains into symbiovar anthyllidis as well as those of M. metallidurans STM2683T, M. delmotii STM4623T and M. prunaredense STM4891T, all recovered from the same legume species.

LIM Homeobox-2 Suppresses Hallmarks of Adult and Pediatric Liver Cancers by Inactivating MAPK/ERK and Wnt/Beta-Catenin Pathways.

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

SMAD4 TGF-ß-independent function preconditions naive CD8+ T cells to prevent severe chronic intestinal inflammation.

SMAD4, a mediator of TGF-ß signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-ß receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-ß signature in naive CD8+ T cells. Mechanistically, prior to TGF-ß signaling, SMAD4 binds to promoters and enhancers of several TGF-ß target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-ß signal, SMAD4 limits the expression of TGF-ß negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-ß. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-ß control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-ß-independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.

PUCHI represses early meristem formation in developing lateral roots of Arabidopsis thaliana.

Lateral root organogenesis is a key process in the development of a plant's root system and its adaptation to the environment. During lateral root formation, an early phase of cell proliferation first produces a four-cell-layered primordium, and only from this stage onwards is a root meristem-like structure, expressing root stem cell niche marker genes, being established in the developing organ. Previous studies reported that the gene regulatory network controlling lateral root formation is organized into two subnetworks whose mutual inhibition may contribute to organ patterning. PUCHI encodes an AP2/ERF transcription factor expressed early during lateral root primordium development and required for correct lateral root formation. To dissect the molecular events occurring during this early phase, we generated time-series transcriptomic datasets profiling lateral root development in puchi-1 mutants and wild types. Transcriptomic and reporter analyses revealed that meristem-related genes were expressed ectopically at early stages of lateral root formation in puchi-1 mutants. We conclude that, consistent with the inhibition of genetic modules contributing to lateral root development, PUCHI represses ectopic establishment of meristematic cell identities at early stages of organ development. These findings shed light on gene network properties that orchestrate correct timing and patterning during lateral root formation.

Tyrosine kinase type A-specific signalling pathways are critical for mechanical allodynia development and bone alterations in a mouse model of rheumatoid arthritis.

ABSTRACT: Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice.

Concurrent BMP Signaling Maintenance and TGF-ß Signaling Inhibition Is a Hallmark of Natural Resistance to Muscle Atrophy in the Hibernating Bear.

Muscle atrophy arises from a multiplicity of physio-pathological situations and has very detrimental consequences for the whole body. Although knowledge of muscle atrophy mechanisms keeps growing, there is still no proven treatment to date. This study aimed at identifying new drivers for muscle atrophy resistance. We selected an innovative approach that compares muscle transcriptome between an original model of natural resistance to muscle atrophy, the hibernating brown bear, and a classical model of induced atrophy, the unloaded mouse. Using RNA sequencing, we identified 4415 differentially expressed genes, including 1746 up- and 2369 down-regulated genes, in bear muscles between the active versus hibernating period. We focused on the Transforming Growth Factor (TGF)-ß and the Bone Morphogenetic Protein (BMP) pathways, respectively, involved in muscle mass loss and maintenance. TGF-ß- and BMP-related genes were overall down- and up-regulated in the non-atrophied muscles of the hibernating bear, respectively, and the opposite occurred for the atrophied muscles of the unloaded mouse. This was further substantiated at the protein level. Our data suggest TGF-ß/BMP balance is crucial for muscle mass maintenance during long-term physical inactivity in the hibernating bear. Thus, concurrent activation of the BMP pathway may potentiate TGF-ß inhibiting therapies already targeted to prevent muscle atrophy.

Impact of the type of endometrial oestrogen preparation for frozen-thawed embryo (vaginal or transdermal) on perinatal outcomes in an artificial cycle.

INTRODUCTION: Serum oestradiol concentration at the time of frozen embryo transfer (FET) in artificial cycle are lower when using transdermal administration of oestrogen for endometrial preparation compared to the vaginal route. This difference could have consequences for placentation and establishment of maternal-foetal circulation. The aim of our study was to compare the birth weight of newborns and the perinatal issues after FET in an artificial cycle with regard to the route of administration of oestrogens. METHODS: Retrospective monocentric cohort study in the medically assisted reproduction department of the University Hospital of Angers, France, between January 2017 and October 2020. Inclusion criteria were age >18 years old and one live birth after FET in an artificial cycle. The main outcome was the birth weight of the newborns. The choice of oestrogens administration (transdermal or vaginal) was left to the patient. RESULTS: 804 FET in artificial cycle were included in our study. Oestrogens were administrated in 356/804(36.6%) patients using transdermal route and in 448/804(45.9%) patients using vaginal route. There were 68/345 (19.1%) live births in the transdermal group and 85/448 (19%) in the vaginal group. There was no difference in the birth weight of the newborns (3320[2100-4165] grams in the transdermal group vs 3327.5[915-4650] grams in the vaginal group, p=0.72). All the other perinatal issues were comparable between the two groups. CONCLUSION: Birth weights and perinatal issues were comparable with regard to the route of administration of oestrogens (vaginal or transdermal) in the context of endometrial preparation before FET in an artificial cycle.

TALC: Transcript-level Aware Long-read Correction.

MOTIVATION: Long-read sequencing technologies are invaluable for determining complex RNA transcript architectures but are error-prone. Numerous 'hybrid correction' algorithms have been developed for genomic data that correct long reads by exploiting the accuracy and depth of short reads sequenced from the same sample. These algorithms are not suited for correcting more complex transcriptome sequencing data. RESULTS: We have created a novel reference-free algorithm called Transcript-level Aware Long-Read Correction (TALC) which models changes in RNA expression and isoform representation in a weighted De Bruijn graph to correct long reads from transcriptome studies. We show that transcript-level aware correction by TALC improves the accuracy of the whole spectrum of downstream RNA-seq applications and is thus necessary for transcriptome analyses that use long read technology. AVAILABILITY AND IMPLEMENTATION: TALC is implemented in C++ and available at https://github.com/lbroseus/TALC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In vitro shared transcriptomic responses of Aedes aegypti to arboviral infections: example of dengue and Rift Valley fever viruses.

BACKGROUND: Arthropod borne virus infections are the cause of severe emerging diseases. Among the diseases due to arboviruses, dengue (DEN) and Rift Valley fever (RVF) are in the top ten in the list of diseases responsible of severe human cases worldwide. Understanding the effects of viral infection on gene expression in competent vectors is a challenge for the development of early diagnostic tools and may enable researchers and policy makers to better anticipate outbreaks in the next future. METHODS: In this study, alterations in gene expression across the entire Aedes aegypti genome during infection with DENV and RVFV were investigated in vitro at two time points of infection, the early phase (24 h) and the late phase (6 days) of infection using the RNA sequencing approach RESULTS: A total of 10 upregulated genes that share a similar expression profile during infection with both viruses at early and late phases of infection were identified. Family B and D clip-domain serine proteases (CLIP) were clearly overrepresented as well as C-type lectins and transferrin. CONCLUSIONS: Our data highlight the presence of 10 viral genes upregulated in Ae. aegypti during infection. They may also be targeted in the case of the development of broad-spectrum anti-viral diagnostic tools focusing the mosquito vectors rather than the mammalian hosts as they may predict the emergence of outbreaks.

Quantifying Genomic Imprinting at Tissue and Cell Resolution in the Brain.

Imprinted genes are a group of ~150 genes that are preferentially expressed from one parental allele owing to epigenetic marks asymmetrically distributed on inherited maternal and paternal chromosomes. Altered imprinted gene expression causes human brain disorders such as Prader-Willi and Angelman syndromes and additional rare brain diseases. Research data principally obtained from the mouse model revealed how imprinted genes act in the normal and pathological brain. However, a better understanding of imprinted gene functions calls for building detailed maps of their parent-of-origin-dependent expression and of associated epigenetic signatures. Here we review current methods for quantifying genomic imprinting at tissue and cell resolutions, with a special emphasis on methods to detect parent-of-origin dependent expression and their applications to the brain. We first focus on bulk RNA-sequencing, the main method to detect parent-of-origin-dependent expression transcriptome-wide. We discuss the benefits and caveats of bulk RNA-sequencing and provide a guideline to use it on F1 hybrid mice. We then review methods for detecting parent-of-origin-dependent expression at cell resolution, including single-cell RNA-seq, genetic reporters, and molecular probes. Finally, we provide an overview of single-cell epigenomics technologies that profile additional features of genomic imprinting, including DNA methylation, histone modifications and chromatin conformation and their combination into sc-multimodal omics approaches, which are expected to yield important insights into genomic imprinting in individual brain cells.

ISoLDE: a data-driven statistical method for the inference of allelic imbalance in datasets with reciprocal crosses.

MOTIVATION: Allelic imbalance (AI), i.e. the unequal expression of the alleles of the same gene in a single cell, affects a subset of genes in diploid organisms. One prominent example of AI is parental genomic imprinting, which results in parent-of-origin-dependent, mono-allelic expression of a limited number of genes in metatherian and eutherian mammals and in angiosperms. Currently available methods for identifying AI rely on data modeling and come with the associated limitations. RESULTS: We have designed ISoLDE (Integrative Statistics of alleLe Dependent Expression), a novel nonparametric statistical method that takes into account both AI and the characteristics of RNA-seq data to infer allelic expression bias when at least two biological replicates are available for reciprocal crosses. ISoLDE learns the distribution of a specific test statistic from the data and calls genes 'allelically imbalanced', 'bi-allelically expressed' or 'undetermined'. Depending on the number of replicates, predefined thresholds or permutations are used to make calls. We benchmarked ISoLDE against published methods, and showed that ISoLDE compared favorably with respect to sensitivity, specificity and robustness to the number of replicates. Using ISoLDE on different RNA-seq datasets generated from hybrid mouse tissues, we did not discover novel imprinted genes (IGs), confirming the most conservative estimations of IG number. AVAILABILITY AND IMPLEMENTATION: ISoLDE has been implemented as a Bioconductor package available at http://bioconductor.org/packages/ISoLDE/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TAG-RNAi overcomes off-target effects in cancer models.

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

Post-production modifications of murine mesenchymal stem cell (mMSC) derived extracellular vesicles (EVs) and impact on their cellular interaction.

In regards to their key role in intercellular communication, extracellular vesicles (EVs) have a strong potential as bio-inspired drug delivery systems (DDS). With the aim of circumventing some of their well-known issues (production yield, drug loading yield, pharmacokinetics), we specifically focused on switching the biological vision of these entities to a more physico-chemical one, and to consider and fine-tune EVs as synthetic vectors. To allow a rational use, we first performed a full physico-chemical (size, concentration, surface charge, cryoTEM), biochemical (western blot, proteomics, lipidomics, transcriptomics) and biological (cell internalisation) characterisation of murine mesenchymal stem cell (mMSC)-derived EVs. A stability study based on evaluating the colloidal behaviour of obtained vesicles was performed in order to identify optimal storage conditions. We evidenced the interest of using EVs instead of liposomes, in regards to target cell internalisation efficiency. EVs were shown to be internalised through a caveolae and cholesterol-dependent pathway, following a different endocytic route than liposomes. Then, we characterised the effect of physical methods scarcely investigated with EVs (extrusion through 50 nm membranes, freeze-drying, sonication) on EV size, concentration, structure and cell internalisation properties. Our extensive characterisation of the effect of these physical processes highlights their promise as loading methods to make EVs efficient delivery vehicles.

Postnatal Tshz3 Deletion Drives Altered Corticostriatal Function and Autism Spectrum Disorder-like Behavior.

BACKGROUND: Heterozygous deletion of the TSHZ3 gene, encoding for the teashirt zinc-finger homeobox family member 3 (TSHZ3) transcription factor that is highly expressed in cortical projection neurons (CPNs), has been linked to an autism spectrum disorder (ASD) syndrome. Similarly, mice with Tshz3 haploinsufficiency show ASD-like behavior, paralleled by molecular changes in CPNs and corticostriatal synaptic dysfunctions. Here, we aimed at gaining more insight into "when" and "where" TSHZ3 is required for the proper development of the brain, and its deficiency crucial for developing this ASD syndrome. METHODS: We generated and characterized a novel mouse model of conditional Tshz3 deletion, obtained by crossing Tshz3flox/flox with CaMKIIalpha-Cre mice, in which Tshz3 is deleted in CPNs from postnatal day 2 to 3 onward. We characterized these mice by a multilevel approach combining genetics, cell biology, electrophysiology, behavioral testing, and bioinformatics. RESULTS: These conditional Tshz3 knockout mice exhibit altered cortical expression of more than 1000 genes, ∼50% of which have their human orthologue involved in ASD, in particular genes encoding for glutamatergic synapse components. Consistently, we detected electrophysiological and synaptic changes in CPNs and impaired corticostriatal transmission and plasticity. Furthermore, these mice showed strong ASD-like behavioral deficits. CONCLUSIONS: Our study reveals a crucial postnatal role of TSHZ3 in the development and functioning of the corticostriatal circuitry and provides evidence that dysfunction in these circuits might be determinant for ASD pathogenesis. Our conditional Tshz3 knockout mouse constitutes a novel ASD model, opening the possibility for an early postnatal therapeutic window for the syndrome linked to TSHZ3 haploinsufficiency.

Both anti-inflammatory and antiviral properties of novel drug candidate ABX464 are mediated by modulation of RNA splicing.

ABX464 is a first-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-inflammatory effects in the DSS-model for inflammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efficacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the effect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-inflammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no effect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like inflammation and HIV infection. The specific dual ability of ABX464 to generate both anti-inflammatory miR-124 and spliced viral RNA may have applicability for the treatment of both inflammatory diseases and HIV infection.

Transcriptomic Analysis With the Progress of Symbiosis in 'Crack-Entry' Legume Arachis hypogaea Highlights Its Contrast With 'Infection Thread' Adapted Legumes.

In root-nodule symbiosis, rhizobial invasion and nodule organogenesis is host controlled. In most legumes, rhizobia enter through infection threads and nodule primordium in the cortex is induced from a distance. But in dalbergoid legumes like Arachis hypogaea, rhizobia directly invade cortical cells through epidermal cracks to generate the primordia. Herein, we report the transcriptional dynamics with the progress of symbiosis in A. hypogaea at 1 day postinfection (dpi) (invasion), 4 dpi (nodule primordia), 8 dpi (spread of infection in nodule-like structure), 12 dpi (immature nodules containing rod-shaped rhizobia), and 21 dpi (mature nodules with spherical symbiosomes). Expression of putative ortholog of symbiotic genes in 'crack entry' legume A. hypogaea was compared with infection thread-adapted model legumes. The contrasting features were i) higher expression of receptors like LYR3 and EPR3 as compared with canonical Nod factor receptors, ii) late induction of transcription factors like NIN and NSP2 and constitutive high expression of ERF1, EIN2, bHLH476, and iii) induction of divergent pathogenesis-responsive PR-1 genes. Additionally, symbiotic orthologs of SymCRK, ROP6, RR9, SEN1, and DNF2 were not detectable and microsynteny analysis indicated the absence of a RPG homolog in diploid parental genomes of A. hypogaea. The implications are discussed and a molecular framework that guides crack-entry symbiosis in A. hypogaea is proposed.

Ma Orthologous Genes in Prunus spp. Shed Light on a Noteworthy NBS-LRR Cluster Conferring Differential Resistance to Root-Knot Nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

The impact of thyroid hormone in seasonal breeding has a restricted transcriptional signature.

Thyroid hormone (TH) directs seasonal breeding through reciprocal regulation of TH deiodinase (Dio2/Dio3) gene expression in tanycytes in the ependymal zone of the medio-basal hypothalamus (MBH). Thyrotropin secretion by the pars tuberalis (PT) is a major photoperiod-dependent upstream regulator of Dio2/Dio3 gene expression. Long days enhance thyrotropin production, which increases Dio2 expression and suppresses Dio3 expression, thereby heightening TH signaling in the MBH. Short days appear to exert the converse effect. Here, we combined endocrine profiling and transcriptomics to understand how photoperiod and TH control the ovine reproductive status through effects on hypothalamic function. Almost 3000 genes showed altered hypothalamic expression between the breeding- and non-breeding seasons, showing gene ontology enrichment for cell signaling, epigenetics and neural plasticity. In contrast, acute switching from a short (SP) to a long photoperiod (LP) affected the expression of a much smaller core of 134 LP-responsive genes, including a canonical group previously linked to photoperiodic synchronization. Reproductive switch-off at the end of the winter breeding season was completely blocked by thyroidectomy (THX), despite a very modest effect on the hypothalamic transcriptome. Only 49 genes displayed altered expression between intact and THX ewes, including less than 10% of the LP-induced gene set. Neuroanatomical mapping showed that many LP-induced genes were expressed in the PT, independently of the TH status. In contrast, TH-sensitive seasonal genes were principally expressed in the ependymal zone. These data highlight the distinctions between seasonal remodeling effects, which appear to be largely independent of TH, and TH-dependent localised effects which are permissive for transition to the non-breeding state.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition.