Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell-death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in testis after sex determination, we have analyzed mice lacking NR5A1 in Sertoli cells (SC) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impairs the expression of genes characteristic of the SC identity (e.g., Sox9, Amh), causes SC death from E14.5 through a Trp53-independent mechanism related to anoikis, and induces disorganization of the testis cords. Together, these effects cause germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SC change their molecular identity: some acquire a "pre-granulosa-like" identity, while other revert to a "supporting progenitor-like" cell identity, most of them being "intersex" because they express both testicular and ovarian genes. Fetal Leydig cells (LC) do not display significant changes, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were absent from the postnatal testes. In addition, adult mutant males display persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which can be explained by the loss of AMH and testosterone synthesis due to SC failure.

Neuronal A2A receptor exacerbates synapse loss and memory deficits in APP/PS1 mice.

Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

Arabidopsis ATRX Modulates H3.3 Occupancy and Fine-Tunes Gene Expression.

Histones are essential components of the nucleosome, the major chromatin subunit that structures linear DNA molecules and regulates access of other proteins to DNA. Specific histone chaperone complexes control the correct deposition of canonical histones and their variants to modulate nucleosome structure and stability. In this study, we characterize the Arabidopsis thaliana Alpha Thalassemia-mental Retardation X-linked (ATRX) ortholog and show that ATRX is involved in histone H3 deposition. Arabidopsis ATRX mutant alleles are viable, but show developmental defects and reduced fertility. Their combination with mutants of the histone H3.3 chaperone HIRA (Histone Regulator A) results in impaired plant survival, suggesting that HIRA and ATRX function in complementary histone deposition pathways. Indeed, ATRX loss of function alters cellular histone H3.3 pools and in consequence modulates the H3.1/H3.3 balance in the cell. H3.3 levels are affected especially at genes characterized by elevated H3.3 occupancy, including the 45S ribosomal DNA (45S rDNA) loci, where loss of ATRX results in altered expression of specific 45S rDNA sequence variants. At the genome-wide scale, our data indicate that ATRX modifies gene expression concomitantly to H3.3 deposition at a set of genes characterized both by elevated H3.3 occupancy and high expression. Together, our results show that ATRX is involved in H3.3 deposition and emphasize the role of histone chaperones in adjusting genome expression.

Blepharocheilodontic syndrome is a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1.

PURPOSE: Blepharocheilodontic (BCD) syndrome is a rare autosomal dominant condition characterized by eyelid malformations, cleft lip/palate, and ectodermal dysplasia. The molecular basis of BCD syndrome remains unknown. METHODS: We recruited 11 patients from 8 families and performed exome sequencing for 5 families with de novo BCD syndrome cases and targeted Sanger sequencing in the 3 remaining families. RESULTS: We identified five CDH1 heterozygous missense mutations and three CTNND1 heterozygous truncating mutations leading to loss-of-function or haploinsufficiency. Establishment of detailed genotype-phenotype correlations was not possible because of the size of the cohort; however, the phenotype seems to appear more severe in case of CDH1 mutations. Functional analysis of CDH1 mutations confirmed their deleterious impact and suggested accelerated E-cadherin degradation. CONCLUSION: Mutations in CDH1 encoding the E-cadherin were previously reported in hereditary diffuse gastric cancer as well as in nonsyndromic cleft lip/palate. Mutations in CTNND1 have never been reported before. The encoded protein, p120ctn, prevents E-cadherin endocytosis and stabilizes its localization at the cell surface. Conditional deletion of Cdh1 and Ctnnd1 in various animal models induces features reminiscent of BCD syndrome and underlines critical role of the E-cadherin-p120ctn interaction in eyelid, craniofacial, and tooth development. Our data assert BCD syndrome as a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1 and widen the phenotypic spectrum of E-cadherin anomalies.Genet Med advance online publication 09 March 2017.

Fast Dating Using Least-Squares Criteria and Algorithms.

Phylogenies provide a useful way to understand the evolutionary history of genetic samples, and data sets with more than a thousand taxa are becoming increasingly common, notably with viruses (e.g., human immunodeficiency virus (HIV)). Dating ancestral events is one of the first, essential goals with such data. However, current sophisticated probabilistic approaches struggle to handle data sets of this size. Here, we present very fast dating algorithms, based on a Gaussian model closely related to the Langley-Fitch molecular-clock model. We show that this model is robust to uncorrelated violations of the molecular clock. Our algorithms apply to serial data, where the tips of the tree have been sampled through times. They estimate the substitution rate and the dates of all ancestral nodes. When the input tree is unrooted, they can provide an estimate for the root position, thus representing a new, practical alternative to the standard rooting methods (e.g., midpoint). Our algorithms exploit the tree (recursive) structure of the problem at hand, and the close relationships between least-squares and linear algebra. We distinguish between an unconstrained setting and the case where the temporal precedence constraint (i.e., an ancestral node must be older that its daughter nodes) is accounted for. With rooted trees, the former is solved using linear algebra in linear computing time (i.e., proportional to the number of taxa), while the resolution of the latter, constrained setting, is based on an active-set method that runs in nearly linear time. With unrooted trees the computing time becomes (nearly) quadratic (i.e., proportional to the square of the number of taxa). In all cases, very large input trees (>10,000 taxa) can easily be processed and transformed into time-scaled trees. We compare these algorithms to standard methods (root-to-tip, r8s version of Langley-Fitch method, and BEAST). Using simulated data, we show that their estimation accuracy is similar to that of the most sophisticated methods, while their computing time is much faster. We apply these algorithms on a large data set comprising 1194 strains of Influenza virus from the pdm09 H1N1 Human pandemic. Again the results show that these algorithms provide a very fast alternative with results similar to those of other computer programs. These algorithms are implemented in the LSD software (least-squares dating), which can be downloaded from http://www.atgc-montpellier.fr/LSD/, along with all our data sets and detailed results. An Online Appendix, providing additional algorithm descriptions, tables, and figures can be found in the Supplementary Material available on Dryad at http://dx.doi.org/10.5061/dryad.968t3.

Searching for virus phylotypes.

MOTIVATION: Large phylogenies are being built today to study virus evolution, trace the origin of epidemics, establish the mode of transmission and survey the appearance of drug resistance. However, no tool is available to quickly inspect these phylogenies and combine them with extrinsic traits (e.g. geographic location, risk group, presence of a given resistance mutation), seeking to extract strain groups of specific interest or requiring surveillance. RESULTS: We propose a new method for obtaining such groups, which we call phylotypes, from a phylogeny having taxa (strains) annotated with extrinsic traits. Phylotypes are subsets of taxa with close phylogenetic relationships and common trait values. The method combines ancestral trait reconstruction using parsimony, with combinatorial and numerical criteria measuring tree shape characteristics and the diversity and separation of the potential phylotypes. A shuffling procedure is used to assess the statistical significance of phylotypes. All algorithms have linear time complexity. This results in low computing times, typically a few minutes for the larger data sets with a number of shuffling steps. Two HIV-1 data sets are analyzed, one of which is large, containing >3000 strains of HIV-1 subtype C collected worldwide, where the method shows its ability to recover known clusters and transmission routes, and to detect new ones. AVAILABILITY: This method and companion tools are implemented in an interactive Web interface (www.phylotype.org), which provides a wide choice of graphical views and output formats, and allows for exploratory analyses of large data sets.

Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors.

Androgen deprivation therapy (ADT) is a cornerstone of prostate cancer (PCa) management. Although tumors initially regress, many progress to a hormone-independent state termed castration-resistant PCa (CRPC), for which treatment options are limited. We here report that the major luminal cell population in tumors of Pten(i)pe-/- mice, generated by luminal epithelial cell-specific deletion of the tumor suppressor PTEN after puberty, is castration-resistant and that the expression of inflammation and stemness markers is enhanced in persistent luminal cells. In addition, hypoxia-inducible factor 1 (HIF1) signaling, which we have previously demonstrated to be induced in luminal cells of Pten(i)pe-/- mice and to promote malignant progression, is further activated. Importantly, we show that genetic and pharmacological inhibition of HIF1A sensitizes Pten-deficient prostatic tumors to castration and provides durable therapeutic responses. Furthermore, HIF1A inhibition induces apoptotic signaling in human CRPC cell lines. Therefore, our data demonstrate that HIF1A in prostatic tumor cells is a critical factor that enables their survival after ADT, and identify it as a target for CRPC management.

RNA polymerase II transcription with partially assembled TFIID complexes.

The recognition of core promoter sequences by the general transcription factor TFIID is the first step in the process of RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Inducible Taf7 knock out (KO) results in the formation of a Taf7-less TFIID complex, while Taf10 KO leads to serious defects within the TFIID assembly pathway. Either TAF7 or TAF10 depletions correlate with the detected TAF occupancy changes at promoters, and with the distinct phenotype severities observed in mouse embryonic stem cells or mouse embryos. Surprisingly however, under either Taf7 or Taf10 deletion conditions, TBP is still associated to the chromatin, and no major changes are observed in nascent Pol II transcription. Thus, partially assembled TFIID complexes can sustain Pol II transcription initiation, but cannot replace holo-TFIID over several cell divisions and/or development.

A time- and space-resolved nuclear receptor atlas in mouse liver.

The functional versatility of the liver is paramount for organismal homeostasis. Adult liver functions are controlled by a tightly regulated transcription factor network including nuclear receptors (NRs), which orchestrate many aspects of hepatic physiology. NRs are transcription factors sensitive to extracellular cues such as hormones, lipids, xenobiotics, etc. and are modulated by intracellular signaling pathways. While liver functional zonation and adaptability to fluctuating conditions rely on a sophisticated cellular architecture, a comprehensive knowledge of NR functions within liver cell populations is still lacking. As a step toward the accurate mapping of NR functions in the liver, we characterized their levels of expression in the whole liver from C57Bl6/J male mice as a function of time and diet. Nr1d1 (Rev-erba), Nr1d2 (Rev-erbb), Nr1c2 (Pparb/d), and Nr1f3 (Rorg) exhibited a robust cyclical expression in ad libitum-fed mice which was, like most cyclically expressed NRs, reinforced upon time-restricted feeding. In a few instances, cyclical expression was lost or gained as a function of the feeding regimen. NR isoform expression was explored in purified hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells, and liver sinusoidal cells. The expression of some NR isoforms, such as Nr1h4 (Fxra) and Nr1b1 (Rara) isoforms, was markedly restricted to a few cell types. Leveraging liver single-cell RNAseq studies yielded a zonation pattern of NRs in hepatocytes, liver sinusoidal cells, and stellate cells, establishing a link between NR subtissular localization and liver functional specialization. In summary, we provide here an up-to-date compendium of NR expression in mouse liver in space and time.

Cell cycle gene regulation dynamics revealed by RNA velocity and deep-learning.

Despite the fact that the cell cycle is a fundamental process of life, a detailed quantitative understanding of gene regulation dynamics throughout the cell cycle is far from complete. Single-cell RNA-sequencing (scRNA-seq) technology gives access to these dynamics without externally perturbing the cell. Here, by generating scRNA-seq libraries in different cell systems, we observe cycling patterns in the unspliced-spliced RNA space of cell cycle-related genes. Since existing methods to analyze scRNA-seq are not efficient to measure cycling gene dynamics, we propose a deep learning approach (DeepCycle) to fit these patterns and build a high-resolution map of the entire cell cycle transcriptome. Characterizing the cell cycle in embryonic and somatic cells, we identify major waves of transcription during the G1 phase and systematically study the stages of the cell cycle. Our work will facilitate the study of the cell cycle in multiple cellular models and different biological contexts.

Istradefylline protects from cisplatin-induced nephrotoxicity and peripheral neuropathy while preserving cisplatin antitumor effects.

Cisplatin is a potent chemotherapeutic drug that is widely used in the treatment of various solid cancers. However, its clinical effectiveness is strongly limited by frequent severe adverse effects, in particular nephrotoxicity and chemotherapy-induced peripheral neuropathy. Thus, there is an urgent medical need to identify novel strategies that limit cisplatin-induced toxicity. In the present study, we show that the FDA-approved adenosine A2A receptor antagonist istradefylline (KW6002) protected from cisplatin-induced nephrotoxicity and neuropathic pain in mice with or without tumors. Moreover, we also demonstrate that the antitumoral properties of cisplatin were not altered by istradefylline in tumor-bearing mice and could even be potentiated. Altogether, our results support the use of istradefylline as a valuable preventive approach for the clinical management of patients undergoing cisplatin treatment.

Radiosensitizing Pancreatic Cancer with PARP Inhibitor and Gemcitabine: An In Vivo and a Whole-Transcriptome Analysis after Proton or Photon Irradiation.

Over the past few years, studies have focused on the development of targeted radiosensitizers such as poly(ADP-ribose) polymerase inhibitors. We performed an in vivo study and a whole-transcriptome analysis to determine whether PARP inhibition enhanced gemcitabine-based chemoradiosensitization of pancreatic cancer xenografts, combined with either proton or photon irradiation. NMRI mice bearing MIA PaCa-2 xenografts were treated with olaparib and/or gemcitabine and irradiated with 10 Gy photon or proton. First, a significant growth inhibition was obtained after 10 Gy proton irradiation compared to 10 Gy photon irradiation (p = 0.046). Moreover, the combination of olaparib, gemcitabine and proton therapy significantly sensitized tumor xenografts, compared to gemcitabine (p = 0.05), olaparib (p = 0.034) or proton therapy (p < 0.0001) alone or to the association of olaparib, gemcitabine and radiotherapy (p = 0.024). Simultaneously, whole RNA sequencing profiling showed differentially expressed genes implicated in categories such as DNA repair, type I interferon signaling and cell cycle. Moreover, a large amount of lncRNA was dysregulated after proton therapy, gemcitabine and olaparib. This is the first study showing that addition of olaparib to gemcitabine-based chemoradiotherapy improved significantly local control in vivo, especially after proton therapy. RNA sequencing profiling analysis presented dynamic alteration of transcriptome after chemoradiation and identified a classifier of gemcitabine response.

Ultrasound and Transcriptomics Identify a Differential Impact of Cisplatin and Histone Deacetylation on Tumor Structure and Microenvironment in a Patient-Derived In Vivo Model of Gastric Cancer.

The reasons behind the poor efficacy of transition metal-based chemotherapies (e.g., cisplatin) or targeted therapies (e.g., histone deacetylase inhibitors, HDACi) on gastric cancer (GC) remain elusive and recent studies suggested that the tumor microenvironment could contribute to the resistance. Hence, our objective was to gain information on the impact of cisplatin and the pan-HDACi SAHA (suberanilohydroxamic acid) on the tumor substructure and microenvironment of GC, by establishing patient-derived xenografts of GC and a combination of ultrasound, immunohistochemistry, and transcriptomics to analyze. The tumors responded partially to SAHA and cisplatin. An ultrasound gave more accurate tumor measures than a caliper. Importantly, an ultrasound allowed a noninvasive real-time access to the tumor substructure, showing differences between cisplatin and SAHA. These differences were confirmed by immunohistochemistry and transcriptomic analyses of the tumor microenvironment, identifying specific cell type signatures and transcription factor activation. For instance, cisplatin induced an "epithelial cell like" signature while SAHA favored a "mesenchymal cell like" one. Altogether, an ultrasound allowed a precise follow-up of the tumor progression while enabling a noninvasive real-time access to the tumor substructure. Combined with transcriptomics, our results underline the different intra-tumoral structural changes caused by both drugs that impact differently on the tumor microenvironment.

Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells.

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.

Chromatin de-condensation by switching substrate elasticity.

Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.

Intragenic FMR1 disease-causing variants: a significant mutational mechanism leading to Fragile-X syndrome.

Fragile-X syndrome (FXS) is a frequent genetic form of intellectual disability (ID). The main recurrent mutagenic mechanism causing FXS is the expansion of a CGG repeat sequence in the 5'-UTR of the FMR1 gene, therefore, routinely tested in ID patients. We report here three FMR1 intragenic pathogenic variants not affecting this sequence, identified using high-throughput sequencing (HTS): a previously reported hemizygous deletion encompassing the last exon of FMR1, too small to be detected by array-CGH and inducing decreased expression of a truncated form of FMRP protein, in three brothers with ID (family 1) and two splice variants in boys with sporadic ID: a de novo variant c.990+1G>A (family 2) and a maternally inherited c.420-8A>G variant (family 3). After clinical reevaluation, the five patients presented features consistent with FXS (mean Hagerman's scores=15). We conducted a systematic review of all rare non-synonymous variants previously reported in FMR1 in ID patients and showed that six of them are convincing pathogenic variants. This study suggests that intragenic FMR1 variants, although much less frequent than CGG expansions, are a significant mutational mechanism leading to FXS and demonstrates the interest of HTS approaches to detect them in ID patients with a negative standard work-up.

Phylodynamics of the major HIV-1 CRF02_AG African lineages and its global dissemination.

The HIV-1 CRF02_AG clade is the most prevalent HIV variant in West and West-Central Africa and its detection outside Africa is increasingly common. Little is known, however, about the number and phylodynamics of major CRF02_AG lineages circulating worldwide. To this end, a total of 3170 HIV-1 CRF02_AG-like pol sequences isolated around the world, over a period of 25years (1989 to 2013), were analyzed using Maximum Likelihood and Bayesian coalescent-based methods. Our results suggest that most of the current CRF02_AG diversity comes from the dissemination of a few founder strains out of Central Africa into West Africa and Cameroon between the late 1960s and the middle 1980s. The CRF02_AG strain introduced into West Africa established a large regional epidemic with low phylogeographic structure. This strain was also successfully disseminated out of the West African region and originated at least three large secondary outbreaks in Cameroon at around the late 1970s, in the former Soviet Union (FSU) countries at around the late 1990s, and in Bulgaria/Germany at around the early 2000s. The CRF02_AG African lineages introduced into Cameroon remained mostly restricted to this country and its neighbors. Demographic reconstructions indicate that major CRF02_AG clades circulating in Africa exhibited a decline in growth rate since the middle 1980s/1990s, whereas CRF02_AG clades in Europe and the FSU countries continue to grow exponentially until the middle to late 2000s. Substantial differences in the median estimated growth rate of the same CRF02_AG clade circulating in different regions (0.63-2.00year-1), and of different CRF02_AG clades circulating in the same country (0.41-0.75year-1) were observed. Thus, the cause of the epidemic outcome of the different HIV-1 CRF02_AG lineages is probably multifactorial.

The origin and molecular epidemiology of HIV.

HIV-1 in humans resulted from at least four cross-species transmissions of simian immunodeficiency viruses (SIVs) from chimpanzees and gorillas in West Central Africa, while HIV-2 viruses resulted from at least eight independent transmissions of SIVs infecting sooty mangabeys in West Africa only, where one of these transmissions (HIV-1 group M) is responsible for the global epidemic. HIV-1 M is subdivided into nine subtypes and a wide diversity of circulating recombinant forms (CRFs) and unique recombinant forms. The heterogenic HIV-1 M subtype/CRF distribution is the result of founder effects. The genetic diversity of HIV-1 continues to increase overtime due to demographic factors such as travel and migration and frequent co/superinfections. In addition, the expanded access to antiretrovirals leads to an increasing number of drug-resistant strains, especially in resource limited countries.