2-hydroxyglutarate (2HG) is recognized as an epigenetic regulator in cancer and in a few physiological states. Of all organs, the testis harbors the highest levels of 2HG, yet it’s putative functions in germ cell biology are unknown. Here we show that 2HG is generated in specific stages of the mouse germ cell lineage by the testis specific lactate dehydrogenase C (LDHC). LDHC is expressed in pachytene, diplotene and diakinesis (PDD) cells and unexpectedly enters nuclei where it localizes along chromosomes and centromeres. LDHC-generated L-2HG controls centromere compaction and pericentromeric heterochromatin organization through multiple effects including clustering of chromocenters, centromere and chromocenter condensation and expression of satellite RNAs. The involvement of L-2HG in the above functions was shown both in isolated PDD cells and in vivo and is specific to the L but not D enantiomer. Our findings reveal that 2HG can rapidly change the conformation of these multisubunit structures and is necessary for the proper progression of the cell cycle.Competing Interest StatementThe authors have declared no competing interest.

INTRODUCTION: As in all vertebrates, reproduction in fish is regulated by GnRH control on gonadotrophic hormones (GtH) activity. However, the neuroendocrine factors that promote GnRH and GtH activity are unknown. In Nile tilapia (Oreochromis niloticus), sexual activity and reproduction ability depend on social rank; only dominant males and females reproduce. Here, this characteristic of dominant fish allows us to compare brain and pituitary gene expression in animals that do and do not reproduce, aiming to reveal mechanisms that regulate reproduction. METHODS: an extensive transcriptome analysis was performed, combining two sets of transcriptomes: a novel whole-brain and pituitary transcriptome of established dominant and subordinate males, together with a cell-specific transcriptome of LH and FSH cells. Pituitary incubation assay validated the direct effect of steroid application on chosen genes and GtH secretion. RESULTS: in most dominant fish, as determined behaviorally, the gonadosomatic index was higher than in subordinate fish, and the leading upregulated pituitary genes were those coding for GtHs. In the brain, various neuropeptide genes, including isotocin, cholecystokinin, and MCH, were upregulated; these may be related to reproductive status through effects on behavior and feeding. In a STRING network analysis combining the two transcriptome sets, brain aromatase, highly expressed in LH cells, is the most central gene with the highest number of connections. In the pituitary incubation assay, testosterone and estradiol increased the secretion of LH and specific gene transcription. CONCLUSIONS: the close correlation between behavioral dominance and reproductive capacity in tilapia allows unraveling novel genes that may regulate the HPG axis, highlighting aromatase as the main factor affecting the brain and pituitary in maintaining a sexually active organism.

Previously, we reported that RelA protein facilitates Hfq-mediated mRNA-sRNA regulation by binding sRNAs carrying a Shine-Dalgarno-like GGAG sequence. In turn, sRNA-Hfq monomers are stabilized, enabling the attachment of more Hfq subunits to form a functional hexamer. Here, using CLIP-seq, we present a global analysis of RelA-bound RNAs showing that RelA interacts with sRNAs as well as with mRNAs carrying a GGAG motif. RelA binding of mRNAs carrying GGAG at position -7 relative to the initiation codon (AUG) inhibits translation by interfering with the binding of 30S ribosomes. The extent of inhibition depends on the distance of GGAG relative to the AUG, as shortening the spacing between GGAG and AUG abrogates RelA-mediated inhibition. Interestingly, RelA binding of target mRNAs carrying GGAG in the coding sequence or close to AUG facilitates target gene regulation by sRNA partners that lack GGAG. However, translation inhibition caused by RelA binding of mRNAs carrying GGAG at position -7 relative to the AUG renders sRNA-mRNA basepairing regulation ineffective. Our study indicates that by binding RNAs carrying GGAG the ribosome-associated RelA protein inhibits translation of specific newly synthesized incoming mRNAs or enables basepairing regulation by their respective sRNA partners, thereby introducing a new regulatory concept for the bacterial response.

A main strategy of bacteria adapting to environmental changes is the remodeling of their transcriptome. Changes in the transcript levels of specific genes are due to combined effects of various regulators, including small RNAs (sRNAs). sRNAs are post-transcriptional regulators of gene expression that mainly control translation, but also directly and indirectly affect the levels of their target transcripts. Yet, the relative contribution of an sRNA to the total change in the transcript level of a gene upon an environmental change has not been assessed. We present a design of differential gene expression analysis by RNA-seq that allows extracting the contribution of an sRNA to the total change in the transcript level of each gene in response to an environmental change by fitting a linear model to the data. We exemplify this for the sRNA RyhB in cells growing under iron limitation and show a variation among genes in the relative contribution of RyhB to the change in their transcript level upon iron limitation, from subtle to very substantial. Extracting the relative contribution of an sRNA to the total change in expression of genes is important for understanding the integration of regulation by sRNAs with other regulatory mechanisms in the cell.

Oogenesis produces functional eggs and is essential for fertility, embryonic development, and reproduction. The zebrafish ovary is an excellent model to study oogenesis in vertebrates, and recent studies have identified multiple regulators in oocyte development through forward genetic screens, as well as reverse genetics by CRISPR mutagenesis. However, many developmental steps in oogenesis, in zebrafish and other species, remain poorly understood, and their underlying mechanisms are unknown. Here, we take a genomic approach to systematically uncover biological activities throughout oogenesis. We performed transcriptomic analysis on five stages of oogenesis, from the onset of oocyte differentiation through Stage III, which precedes oocyte maturation. These transcriptomes revealed thousands of differentially expressed genes across stages of oogenesis. We analyzed trends of gene expression dynamics along oogenesis, as well as their expression in pair-wise comparisons between stages. We determined their functionally enriched terms, identifying uniquely characteristic biological activities in each stage. These data identified two prominent developmental phases in oocyte differentiation and traced the accumulation of maternally deposited embryonic regulator transcripts in the developing oocyte. Our analysis provides the first molecular description for oogenesis in zebrafish, which we deposit online as a resource for the community. Further, the presence of multiple gene paralogs in zebrafish, and the exclusive curation by many bioinformatic tools of the single paralogs present in humans, challenge zebrafish genomic analyses. We offer an approach for converting zebrafish gene name nomenclature to the human nomenclature for supporting genomic analyses generally in zebrafish. Altogether, our work provides a valuable resource as a first step to uncover oogenesis mechanisms and candidate regulators and track accumulating transcripts of maternal regulators of embryonic development.

The blood-brain barrier (BBB) selectively regulates the entry of molecules into the central nervous system (CNS). A crosstalk between brain microvascular endothelial cells (BMECs) and resident CNS cells promotes the acquisition of functional tight junctions (TJs). Retinoic acid (RA), a key signaling molecule during embryonic development, is used to enhance in vitro BBB models' functional barrier properties. However, its physiological relevance and affected pathways are not fully understood. P450 oxidoreductase (POR) regulates the enzymatic activity of microsomal cytochromes. POR-deficient (PORD) patients display impaired steroid homeostasis and cognitive disabilities. Here, we used both patient-specific POR-deficient and CRISPR-Cas9-mediated POR-depleted induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) to study the role of POR in the acquisition of functional barrier properties. We demonstrate that POR regulates cellular RA homeostasis and that POR deficiency leads to the accumulation of RA within iBMECs, resulting in the impaired acquisition of TJs and, consequently, to dysfunctional development of barrier properties.

We recently identified a missense mutation in Nucleoporin107 (Nup107; D447N) underlying XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells.

BACKGROUNDCytomegalovirus (CMV) is the most common intrauterine infection, leading to infant brain damage. Prognostic assessment of CMV-infected fetuses has remained an ongoing challenge in prenatal care, in the absence of established prenatal biomarkers of congenital CMV (cCMV) infection severity. We aimed to identify prognostic biomarkers of cCMV-related fetal brain injury.METHODSWe performed global proteome analysis of mid-gestation amniotic fluid samples, comparing amniotic fluid of fetuses with severe cCMV with that of asymptomatic CMV-infected fetuses. The levels of selected differentially excreted proteins were further determined by specific immunoassays.RESULTSUsing unbiased proteome analysis in a discovery cohort, we identified amniotic fluid proteins related to inflammation and neurological disease pathways, which demonstrated distinct abundance in fetuses with severe cCMV. Amniotic fluid levels of 2 of these proteins - the immunomodulatory proteins retinoic acid receptor responder 2 (chemerin) and galectin-3-binding protein (Gal-3BP) - were highly predictive of the severity of cCMV in an independent validation cohort, differentiating between fetuses with severe (n = 17) and asymptomatic (n = 26) cCMV, with 100%-93.8% positive predictive value, and 92.9%-92.6% negative predictive value (for chemerin and Gal-3BP, respectively). CONCLUSIONAnalysis of chemerin and Gal-3BP levels in mid-gestation amniotic fluids could be used in the clinical setting to profoundly improve the prognostic assessment of CMV-infected fetuses.FUNDINGIsrael Science Foundation (530/18 and IPMP 3432/19); Research Fund - Hadassah Medical Organization.

The virulence of Plasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites "choose" to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression between var genes, each encoding a different PfEMP1 variant. While the mechanisms that regulate mutually exclusive expression of var genes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the active var gene were implicated in the "choice" of the single active var gene. Here, we show that this lncRNA colocalizes with the site of var mRNA transcription and is anchored to the var locus via DNA:RNA interactions. We define the var lncRNA interactome and identify a redox sensor, P. falciparum thioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with the var antisense lncRNA. We show that PfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, when var transcription occurs. In addition, PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation of var2csa, similar to overexpression of PfSAMS. Furthermore, we show that PfTPx-1 knockdown alters the var switch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclear PfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.

Cytochrome c oxidase (COX), a multimeric protein complex, is the final electron acceptor in the mitochondrial electron transfer chain. Primary COX deficiency, caused by mutations in either mitochondrial DNA or nuclear-encoded genes, is a heterogenous group of mitochondrial diseases with a wide range of presentations, ranging from fatal infantile to subtler. We previously reported a patient with primary COX deficiency due to a pathogenic variant in COX4I1 (encoding the common isoform of COX subunit 4, COX4-1), who presented with bone marrow failure, genomic instability, and short stature, mimicking Fanconi anemia (FA). In the present study, we demonstrated that accumulative DNA damage coincided primarily with proliferative cells in the patient's fibroblasts and in COX4i1 knockdown cells. Expression analysis implicated a reduction in DNA damage response pathways, which was verified by demonstrating impaired recovery from genotoxic insult and decreased DNA repair. The premature senescence of the COX4-1-deficient cells prevented us from undertaking additional studies; nevertheless, taken together, our results indicate replicative stress and impaired nuclear DNA damage response in COX4-1 deficiency. Interestingly, our in vitro findings recapitulated the patient's presentation and present status.

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.

BACKGROUND: The dorsal domain of the neural tube is an excellent model to investigate the generation of complexity during embryonic development. It is a highly dynamic and multifaceted region being first transiently populated by prospective neural crest (NC) cells that sequentially emigrate to generate most of the peripheral nervous system. Subsequently, it becomes the definitive roof plate (RP) of the central nervous system. The RP, in turn, constitutes a patterning center for dorsal interneuron development. The factors underlying establishment of the definitive RP and its segregation from NC and dorsal interneurons are currently unknown. RESULTS: We performed a transcriptome analysis at trunk levels of quail embryos comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled molecular heterogeneity between NC and RP stages, and within the RP itself. By implementing these genes, we asked whether Notch signaling is involved in RP development. First, we observed that Notch is active at the RP-interneuron interface. Furthermore, gain and loss of Notch function in quail and mouse embryos, respectively, revealed no effect on early NC behavior. Constitutive Notch activation caused a local downregulation of RP markers with a concomitant development of dI1 interneurons, as well as an ectopic upregulation of RP markers in the interneuron domain. Reciprocally, in mice lacking Notch activity, both the RP and dI1 interneurons failed to form and this was associated with expansion of the dI2 population. CONCLUSIONS: Collectively, our results offer a new resource for defining specific cell types, and provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1(-/-)) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.

Long intergenic non-coding RNAs (lincRNAs) are transcripts longer than 200 nucleotides that are transcribed from non-coding loci yet undergo biosynthesis similar to coding mRNAs. The disproportional number of lincRNAs expressed in testes suggests that lincRNAs are important during gametogenesis, but experimental evidence has implicated very few lincRNAs in this process. We took advantage of the relatively limited number of lincRNAs in the genome of the nematode Caenorhabditis elegans to systematically analyse the functions of lincRNAs during meiosis. We deleted six lincRNA genes that are highly and dynamically expressed in the C. elegans gonad and tested the effects on central meiotic processes. Surprisingly, whereas the lincRNA deletions did not strongly impact fertility, germline apoptosis, crossovers, or synapsis, linc-4 was required for somatic growth. Slower growth was observed in linc-4-deletion mutants and in worms depleted of linc-4 using RNAi, indicating that linc-4 transcripts are required for this post-embryonic process. Unexpectedly, analysis of worms depleted of linc-4 in soma versus germline showed that the somatic role stems from linc-4 expression in germline cells. This unique feature suggests that some lincRNAs, like some small non-coding RNAs, are required for germ-soma interactions.

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.