Proximal Element Research Articles

Retroposition of the Long Transcript from Multiexon IFN-β Homologs in Ancestry Vertebrate Gave Rise to the Proximal Transcription Elements of Intronless IFN-β Promoter in Humans.

IFN-β is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-β and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-β in chicken and humans.

Bone histology of Broad-snouted Caiman Caiman latirostris (Crocodylia: Alligatoridae) as tool for morphophysiological inferences in Crocodylia

Bone histology is an important tool for the interpretation of life patterns in animals of the past and extant fauna. The crocodylians have been studied as important inferential models for morphophysiological characteristics. We aimed to characterize the osteohistology of captive Caiman latirostris, identifying its microanatomy related to growth rates, ontogeny, and environmental conditions. We analyzed five pairs of humeri (proximal elements of the appendicular skeleton) and ribs (axial skeleton) of females’ caiman. Ribs showed, in general, woven-fibered tissues, with low vascularization and parallel-fibered bone and many resorption and erosion cavities. It presented lines of arrested growth (LAGs) in three individuals, without skeletochronological compatibility. Humeri showed a gradient of woven-fibered to parallel-fibered and lamellar-zonal bone as the individuals aging. We observed compacted coarse cancellous bone (CCCB) and a higher number of LAGs in older specimens. Ribs remodel faster than humerus, showing an intra-individual histovariability. The humeri indicated an evident growth pattern with different ontogeny stages and growth rates in different ages. Fast-growing tissues are uncommon in crocodylians, but basal metabolism and optimal growth conditions can lead to this. Bone histology of C. latirostris shows patterns that can be used as inferential models for extant and extinct groups, but we encourage further studies for a better understanding, under different environmental conditions, such as temperature and food availability.

Characterization of the nutrient composition of German beer styles for the German nutrient database

Scientific data of nutrient variations in different beer styles are scarce. This study focuses on characterizing German beer styles in regard to their differences in nutrient composition and aims to improve available data for the calculation of nutritional value. 25 retail beer samples of five different German beer styles (Pilsner beer, wheat beer, crystal wheat beer, export beer, dark beer) were analyzed for their nutrient content. Overall this survey revealed significant differences (p<0.05) in the proximate composition (ash, protein, ethanol, glucose), vitamins (thiamine, riboflavin, vitamin B6) and elements (copper, manganese) for the chosen beer styles. The benefit of these new data is exemplified by the calculation of the nutritional supply for average beer consumption and compared with previous data. For the average beer consumer, this study gives higher values for the percentage of daily nutrient recommendation of niacin, vitamin B6 and folate, whereas lower values are given for alcohol and elements like copper and iodine. Our findings demonstrate that these new data provide an improved basis for nutritional value calculation in future consumption studies.

ANALYSIS OF THE STRESS-STRAIN STATE THREE-DIMENSIONAL MODEL OF A HEALTHY SHOULDER JOINT

Objective. To work out as close as possible to normal human anatomy three-dimensional finite element model of the shoulder joint with elastic ligaments as well as with muscles and the spatial location of their attachment points, to analyze the stress-strain state of the element proximal humerus and scapula. Methods. A geometric model of the humerus and scapulae are constructed. The three-dimensional modeling of the shoulder join based on the geometric models was used with software SolidWorks with mathematical modeling method finite elements and the stress-strain state analysis in the applicationpackage Ansys software. To approach the real conditions of the model we have added the elastic elements that mimic muscles. Model loaded with forces that reproduce the effort in the muscles, applied to the respective contact planes on the humerus head of the human bone. The stress-strain state of proximal elements is calculated in the humerus and scapula for the angles of the abduction — 0 °, 30°, 60° and 90° in neutral rotation of the humerus.Results. The tensile stresses in the scapula are distributed in such a way that at an angle of 0 ° the limb is not raised +5.67 MPa in the area below the joint depressions. The minimum values of the compressive stress have been reached 18.5 MPa. Maximum stresses are in 1.5–2 times higher area of the articular cartilage of the humerus head compared to the cartilage of the glenoid cavity of the scapula. It is established that the dependence of the valuesof the area of the contact zone in the range of change limb abduction angle (0° ... 90°) can be approximated section of a cubic parabola, with changes in area insignificant and are equal to +2.26% — 7.3 % of the value in neutral position at an angle of 0°. Minor differences with the results of similar studies indicate that the validity of the developed mathematical model. Conclusions. The proposed model would allow performing more correct mathematical modeling and comparative analysis of the stress-strain state for various methods of surgical treatment of pathology shoulder joint, in particular arthroplasty.

From the Editor’s desk…

From the Editor’s desk…

The Effect of Impregnation Ratio on the Surface Characteristics of Gigantochloa Verticillata Bamboo-Activated Carbon

The activation process is the final stage in the manufacturing of activated carbon that can be carried out physically or chemically. This paper focuses on characterizing the surface properties of activated carbons from Gigantochloa verticillata bamboo that are chemically activated at 750°C under different impregnation ratios (IRs) of 1:1, 2:1, and 3:1. The activated carbons produced were denoted as IR1-AC, IR2-AC, and IR3-AC for impregnation ratios of 1:1, 2:1, and 3:1, respectively. Characterizations include TGA, SEM, and adsorption isotherm tests. The results of the research show that variation of the impregnation ratio yielded fluctuated content of proximate elements and surface properties of activated carbons. The highest fixed carbon of 75.69% and the lowest ash of 13.10% were obtained by IR2-AC. The highest surface area of 511.10 m2/g and pore volume of 0.561 cc/g was obtained by IR3-AC and IR2-AC, respectively. The activated carbon pores are distributed in micropores and mesopores areas with average pore diameters of 1.245, 2.494, and 1.984 nm for IR1-AC, IR2-AC, and IR3-AC, respectively. The existence of the pores can be found on the surface morphology of activated carbons.

In silico Characterization of the Heme Oxygenase 1 From Bottlenose Dolphin (Tursiops truncatus): Evidence of Changes in the Active Site and Purifying Selection.

Cetacea is a clade well-adapted to the aquatic lifestyle, with diverse adaptations and physiological responses, as well as a robust antioxidant defense system. Serious injuries caused by boats and fishing nets are common in bottlenose dolphins (Tursiops truncatus); however, these animals do not show signs of serious infections. Evidence suggests an adaptive response to tissue damage and associated infections in cetaceans. Heme oxygenase (HO) is a cytoprotective protein that participates in the anti-inflammatory response. HO catalyzes the first step in the oxidative degradation of the heme group. Various stimuli, including inflammatory mediators, regulate the inducible HO-1 isoform. This study aims to characterize HO-1 of the bottlenose dolphin in silico and compare its structure to the terrestrial mammal protein. Upstream HO-1 sequence of the bottlenose dolphin was obtained from NCBI and Ensemble databases, and the gene structure was determined using bioinformatics tools. Five exons and four introns were identified, and proximal regulatory elements were detected in the upstream region. The presence of 10 α-helices, three 310 helices, the heme group lodged between the proximal and distal helices, and a histidine-25 in the proximal helix serving as a ligand to the heme group were inferred for T. truncatus. Amino acid sequence alignment suggests HO-1 is a conserved protein. The HO-1 “fingerprint” and histidine-25 appear to be fully conserved among all species analyzed. Evidence of positive selection within an α-helix configuration without changes in protein configuration and evidence of purifying selection were found, indicating evolutionary conservation of the coding sequence structure.

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of transcription factors NF-Y to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

ABSTRACTSARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Novel function of SART1 in HNF4α transcriptional regulation contributes to its antiviral role during HBV infection

Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and invivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element. We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA. Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.

Identification of Nrf2-responsive microRNA networks as putative mediators of myocardial reductive stress

Although recent advances in the treatment of acute coronary heart disease have reduced mortality rates, few therapeutic strategies exist to mitigate the progressive loss of cardiac function that manifests as heart failure. Nuclear factor, erythroid 2 like 2 (Nfe2l2, Nrf2) is a transcriptional regulator that is known to confer transient myocardial cytoprotection following acute ischemic insult; however, its sustained activation paradoxically causes a reductive environment characterized by excessive antioxidant activity. We previously identified a subset of 16 microRNAs (miRNA) significantly diminished in Nrf2-ablated (Nrf2−/−) mouse hearts, leading to the hypothesis that increasing levels of Nrf2 activation augments miRNA induction and post-transcriptional dysregulation. Here, we report the identification of distinct miRNA signatures (i.e. “reductomiRs”) associated with Nrf2 overexpression in a cardiac-specific and constitutively active Nrf2 transgenic (caNrf2-Tg) mice expressing low (TgL) and high (TgH) levels. We also found several Nrf2 dose-responsive miRNAs harboring proximal antioxidant response elements (AREs), implicating these “reductomiRs” as putative meditators of Nrf2-dependent post-transcriptional regulation. Analysis of mRNA-sequencing identified a complex network of miRNAs and effector mRNAs encoding known pathological hallmarks of cardiac stress-response. Altogether, these data support Nrf2 as a putative regulator of cardiac miRNA expression and provide novel candidates for future mechanistic investigation to understand the relationship between myocardial reductive stress and cardiac pathophysiology.

Assisting translations in border crossing

Abstract This contribution explores the position of translation policy and its connection to translation flows in translation studies research. Power relationships (among languages, cultures, etc.) are essential when assessing and interpreting translation flows. Despite the hyper-centrality of English, other countries and language areas develop cultural policies to export their own literature through translation. The case study furnished here deals with the state agents responsible for the literary translation policy of Estonia, viz., the Estonian Literature Centre and the Traducta translation grants of the Estonian Cultural Endowment (Eesti Kultuurkapital). The statutes of the Traducta program, including the selection criteria, express an instrumental view on translation that is partly in conflict with the findings of modern translation studies. The data on the awards of the Traducta program not only confirm that the publishing of a translation often depends on the additional financial support for the program, but also highlight the significant differences between different target language areas, in a complex interplay of economic factors with elements of cultural image-building and geographical proximity.

B Lymphocyte Specification Is Preceded by Extensive Epigenetic Priming in Multipotent Progenitors.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

Identification of in vivo Hox13-binding sites reveals an essential locus controlling zebrafish brachyury expression.

During early embryogenesis, the vertebrate embryo extends from anterior to posterior because of the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for NMps to retain mesodermal potential and, hence, normal axis development. We recently showed that Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor-binding sites, we identified more than 500 potential Hox13 target genes in zebrafish. Importantly, we found two highly conserved Hox13-binding elements far from the tbxta transcription start site that also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis-stage expression to a tbxta promoter that, on its own, only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.

Rapid sequence evolution driven by transposable elements at a virulence locus in a fungal wheat pathogen

BackgroundPlant pathogens cause substantial crop losses in agriculture production and threaten food security. Plants evolved the ability to recognize virulence factors and pathogens have repeatedly escaped recognition due rapid evolutionary change at pathogen virulence loci (i.e. effector genes). The presence of transposable elements (TEs) in close physical proximity of effector genes can have important consequences for gene regulation and sequence evolution. Species-wide investigations of effector gene loci remain rare hindering our ability to predict pathogen evolvability.ResultsHere, we performed genome-wide association studies (GWAS) on a highly polymorphic mapping population of 120 isolates of Zymoseptoria tritici, the most damaging pathogen of wheat in Europe. We identified a major locus underlying significant variation in reproductive success of the pathogen and damage caused on the wheat cultivar Claro. The most strongly associated locus is intergenic and flanked by genes encoding a predicted effector and a serine-type endopeptidase. The center of the locus contained a highly dynamic region consisting of multiple families of TEs. Based on a large global collection of assembled genomes, we show that the virulence locus has undergone substantial recent sequence evolution. Large insertion and deletion events generated length variation between the flanking genes by a factor of seven (5–35 kb). The locus showed also strong signatures of genomic defenses against TEs (i.e. RIP) contributing to the rapid diversification of the locus.ConclusionsIn conjunction, our work highlights the power of combining GWAS and population-scale genome analyses to investigate major effect loci in pathogens.

Cohesin subunit Rad21 binds to the HSV-1 genome near CTCF insulator sites during latency in vivo.

Herpes Simplex Virus 1 (HSV-1) is a human pathogen that has the ability to establish a lifelong infection in the host. During latency, HSV-1 genomes are chromatinized and are abundantly associated with histones in sensory neurons, yet the mechanisms that govern the latent-lytic transition remain unclear. We hypothesize that the latent-lytic switch is controlled by CTCF insulators, positioned within the HSV-1 latent genome. CTCF insulators, together with the cohesin complex, have the ability to establish and maintain chromtin loops that allow distance separated gene regions to be spatially oriented for transcriptional control. In this current study, we demonstrated that the cohesin subunit Rad21 was recruited to latent HSV-1 genomes near four of the CTCF insulators during latency. We showed that the CTCF insulator known as CTRS1/2, positioned downstream from the essential transactivating IE region of ICP4 was only enriched in Rad21 prior to but not during latency, suggesting that the CTRS1/2 insulator is not required for the maintenance of latency. Further, deletion of the CTRL2 insulator, positioned downstream from the LAT enhancer, resulted in a loss of Rad21 enrichment at insulators flanking the ICP4 region at early times post-infection in mice ganglia, suggesting that these insulators are interdependent. Finally, deletion of the CTRL2 insulator resulted in a loss of Rad21 enrichment at the CTRL2 insulator in a cell-type specific manner, and this loss of Rad21 enrichment was correlated to decreased LAT expression, suggesting that Rad21 recruitment to viral genomes is important for efficient gene expression.ImportanceCTCF insulators are important for transcriptional control and increasing evidence suggests that that CTCF insulators, together with the cohesin complex, regulate viral transcription in DNA viruses. The CTCF-cohesin interaction is important for the formation of chromatin loops, structures that orient distance separated elements in close spatial proximity for transcriptional control. Herpes Simplex Virus 1 (HSV-1) has seven putative CTCF insulators that flank the LAT and the IE, indicating that CTCF insulators play a role in the transition from latency to reactivation. Contributions from the work presented here include the finding that CTCF insulators in HSV-1 genomes are differentially enriched in the cohesin subunit Rad21, suggesting that CTCF-cohesin interactions could be establishing and anchoring chromatin loop structures to control viral transcription.

Morphological divergence, tail loss, and predation risk in urban lizards

Urbanisation changes the structure of natural habitats and alters trophic interactions, pressuring urban species to adjust and adapt. Unlike behavioural shifts that tend to be more flexible, morphological characteristics in vertebrates are less plastic and changes may come at a greater cost. We explored the hypothesis that morphological characteristics in a tropical agamid lizard Psammophilus dorsalis are not only selected to suit altered habitat conditions in urbanised areas but reflect predation risk in urban habitats. In addition, we explored differences in tail loss in both sexes from urban and rural habitats. First, we find that predation risk did not differ between urban and rural habitats, but differed between sexes. Conspicuous courtship-coloured male lizards were more likely to be attacked compared to cryptically-coloured female lizards. Next, we find that P. dorsalis differed in key morphological characteristics, with greater urban-rural differences in males than females. Urban lizards had shorter proximal limb elements, longer 4th digits and greater body mass than rural lizards; and among males, urban lizards had significantly larger head sizes and longer distal limb elements than rural lizards. Finally, we find that the rates of tail loss differed between habitats and sexes, with urban male lizards having greater rates of tail loss than rural males and all females. Inter-sexual difference in tail loss was consistent with the difference in predation risk between sexes, but tail loss differences between urban and rural habitats could not be explained by predation risk. In sum, we find that adaptations to novel urban environments are not exclusively driven by a single promoter (e.g. predation risk) but are influenced by multiple selection pressures with associated fitness costs, such as tail loss.

An XFEM multilayered heaviside enrichment for fracture propagation with reduced enhanced degrees of freedom

SummaryThe development of advanced numerical techniques such as eXtended/Generalized Finite Elements Methods (XFEM/GFEM) has provided means for accurate prediction of material failure. However, the present theories mostly rely on a global formulation, where the system of equations is subject to progressive dimension increase with crack evolution. In this regard, an independent multilayered enrichment is proposed for the XFEM/GFEM family of methods where a few elements in close proximity are assigned to an enrichment layer independent of the remaining ones. The enhanced degrees of freedom can be condensed out at the layer level, which leads to system dimensions, sparsity, and bandness identical to those of the underlying finite elements. Nodal and elemental enrichment methods are shown to be particular limit cases of present approach. The robustness of the proposed approach is first demonstrated in element‐level examples. The use of only few adjacent elements in a group enrichment is shown to suffice for acceptable results while the order of the condition number of the final stiffness matrix resembles the underlying uncracked finite element counterpart. Finally, using several structural examples, the accuracy and robustness of the method is shown in terms of force–displacement response, stress fields, and traction continuity in nonlinear problems.

Mutation of the GnRHR Proximal Promoter AP-1 Element in Mice Results in Suboptimal GnRH Induction of LH and an Abnormal Reproductive Phenotype

Reproduction is regulated by the gonadotropins, LH and FSH, which are synthesized and secreted by pituitary gonadotrophs in response to hypothalamic GnRH in a pulse frequency dependent manner. The gonadotroph decodes GnRH pulsatility via the GnRH receptor (GnRHR), which increases in expression and cell surface density before estrus and is responsible for downstream signaling cascades that differentially favor gonadotropin expression. The gonadotroph Gnrhr promoter contains a tripartite enhancer, including an AP-1 element that is necessary for full GnRH induction of Gnrhr expression in vitro. We previously generated an AP-1 knock-in (KI) mouse model with a single point mutation (C-269T) in the Gnrhr promoter AP-1 binding motif that resulted in an abnormal reproductive phenotype in female mice. Compared to wildtype (WT) littermates, female KI mice had a significant delay in first estrus, disrupted estrous cyclicity, fewer corpora lutea, and smaller litters. Males had no apparent reproductive phenotype. Basal serum gonadotropin levels were similar between WT and KI mice, but gonadectomy induced a significantly lower rise in serum LH levels of KI mice relative to WT mice, concomitant with significantly lower pituitary Gnrhr, Lhb, and Fshb mRNA levels in both sexes. We have now extended the characterization of these mice by measuring LH pulsatility and assessing GnRH induction of LH in vivo and in vitro. The frequency and amplitude of LH pulses over three hours were similar in ovariectomized WT and KI mice; however, KI mice had significantly reduced LH secretion, as measured by area under the curve. Similarly, GnRH treatment induced a diminished LH response in intact KI compared to WT males. In vitro cultures of hemi-pituitaries from gonadectomized WT and KI males were exposed to 0.01 nM GnRH and LH secretion into culture media was measured by ELISA at 0, 0.5, 1, 2, and 4 hours. There was no difference in basal LH secretion between WT and KI pituitaries but GnRH induction of LH was significantly lower in cultures from AP-1 mutant mice, indicating a direct impairment of GnRH action at the level of the pituitary. Taken together, these data indicate that the gonadotroph Gnrhr AP-1 promoter motif is critical for normal reproductive function. Prevention of AP-1 binding to the Gnrhr proximal promoter element decreases GnRH-induced Gnrhr, Lhb, and Fshb levels, impairs GnRH-stimulated LH secretion, and disrupts pubertal development and reproductive cyclicity in female mice.

Chromosome level reference of Atlantic halibut Hippoglossus hippoglossus provides insight into the evolution of sexual determination systems.

Changes in the genetic mechanisms that control sexual determination have occurred independently across the tree of life, and with exceptional frequency in teleost fishes. To investigate the genomic changes underlying the evolution of sexual determination, we sequenced a chromosome-level genome, multitissue transcriptomes, and reduced representation population data for the Atlantic halibut (Hippoglossus hippoglossus), which has an XY/XX sex determination mechanism and has recently diverged (0.9-3.8Ma) from the Pacific halibut (Hippoglossus stenolepis), which has a ZZ/ZW system. We used frequency and coverage-based population approaches to identify a putative sex-determining factor, GSDF. We characterized regions with elevated heterozygosity and linkage disequilibrium indicating suppression of recombination across a nascent sex chromosome. We detected testis-specific expression of GSDF, the sequence of which is highly conserved across flatfishes. Based on evidence from genome-wide association, coverage, linkage disequilibrium, testis and brain transcriptomes, and sequence conservation with other flatfishes, we propose a mechanism for the recent evolution of an XY sex-determination mechanism in Atlantic halibut. Changes to the ancestral sex-determining gene DMRT1 in regulating the downstream gene GSDF probably coincided with GSDF, or a proximal regulatory element of it, becoming the primary sex-determining factor. Our results suggest changes to a small number of elements can have drastic repercussions for the genomic substrate available to sex-specific evolutionary forces, providing insight into how certain elements repeatedly evolve to control sex across taxa. Our chromosome-level assembly, multitissue transcriptomes, and population genomic data provide a valuable resource and understanding of the evolution of sexual systems in fishes.