The initial aim of this study on Balb/C mice was to investigate the putative effects on feeding and appetite of isopentenyl pyrophosphate (IPP) and E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), also known as phosphoantigens (pAgs). HMBPP was recently shown to increase blood meal appetite in malaria mosquitoes. Both IPP and HMBPP are metabolites produced by the normal gut microbiota and apicomplexan parasites such as Plasmodium. To explore potential effects on appetite, male and female mice were treated by gavage with these metabolites, and body mass and gene expression were monitored in brain, stomach and small intestine at 3 h and 7 weeks. Body mass gain did not clearly differ between pAg-treated and water control mice. However, beginning between 4 and 7 weeks, the salivary glands of IPP-treated males began to swell. With the autoimmune Sjögren disease (SjD) in mind, we subsequently investigated the salivary glands after 1, 4 and 7 weeks of IPP treatment. Fast gene set enrichment analysis (FGSEA) of marginal zone B-cell (MZB) transcripts from salivary glands, together with B-cell infiltration in both sexes at 4 weeks, suggested similarities to SjD pathology. Using ELISA, we measured serum autoantibodies against Ro52, Ro60 and La. Multivariate analysis at 7 weeks showed treatment-associated trends: levels of anti-Ro52 and anti-La tended to increase in IPP-treated males, but not in females. Notably, IL-6 serum levels displayed a sex-dependent pattern, and PCA analyses of transcriptomic data from brain, stomach and small intestine—though with some exceptions—also indicated differential responses to pAgs between males and females.

Mononuclear phagocytes facilitate the dissemination of the obligate intracellular parasite Toxoplasma gondii. Here, we report how a set of secreted parasite effector proteins from dense granule organelles (GRA) orchestrates dendritic cell-like chemotactic and pro-inflammatory activation of parasitized macrophages. These effects enabled efficient dissemination of the type II T. gondii lineage, a highly prevalent genotype in humans. We identify novel functions for effectors GRA15 and GRA24 in promoting CCR7-mediated macrophage chemotaxis by acting on NF-κB and p38 mitogen-activated protein kinase signaling pathways, respectively, with contributions by GRA16/18 and counter-regulation by effector TEEGR. Furthermore, GRA28 boosted chromatin accessibility and GRA15/24/NF-κB-dependent transcription at the Ccr7 gene locus in primary macrophages. In vivo, adoptively transferred macrophages infected with wild-type T. gondii outcompeted macrophages infected with a GRA15/24 double mutant in migrating to secondary organs in mice. The data show that T. gondii, rather than being passively shuttled, actively promotes its dissemination by inducing a finely regulated pro-migratory state in parasitized human and murine phagocytes via co-operating polymorphic GRA effectors.

In male mice, the transcription factor A-MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A-MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A-MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiate the production of piRNAs from A-MYB-dependent older loci, ensuring the self-propagation of pachytene piRNAs. A-MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals.

CRISPR/Cas9-driven cancer modeling studies are based on the disruption of tumor suppressor genes by small insertions or deletions (indels) that lead to frame-shift mutations. In addition, CRISPR/Cas9 is widely used to define the significance of cancer oncogenes and genetic dependencies in loss-of-function studies. However, how CRISPR/Cas9 influences gain-of-function oncogenic mutations is elusive. Here, we demonstrate that single guide RNA targeting exon 3 of Ctnnb1 (encoding β-catenin) results in exon skipping and generates gain-of-function isoforms in vivo. CRISPR/Cas9-mediated exon skipping of Ctnnb1 induces liver tumor formation in synergy with YAP^S127A in mice. We define two distinct exon skipping-induced tumor subtypes with different histological and transcriptional features. Notably, ectopic expression of two exon-skipped β-catenin transcript isoforms together with YAP^S127A phenocopies the two distinct subtypes of liver cancer. Moreover, we identify similar CTNNB1 exon-skipping events in patients with hepatocellular carcinoma. Collectively, our findings advance our understanding of β-catenin-related tumorigenesis and reveal that CRISPR/Cas9 can be repurposed, in vivo, to study gain-of-function mutations of oncogenes in cancer. 

Male gametogenesis, spermatogenesis, is a stepwise developmental process to generate mature sperm. The most intricate process of spermatogenesis is meiosis during which two successive cell divisions ensue with dramatic cellular and molecular changes to produce haploid cells. After entry into meiosis, several forms of regulatory events control the orderly progression of meiosis and the timely entry into post-meiotic sperm differentiation. Among other mechanisms, changes to gene expression are controlled by key transcription factors. In this review, we will discuss the gene regulatory mechanisms underlying meiotic entry, meiotic progression, and post-meiotic differentiation with a particular emphasis on the MYBL1/TCFL5 regulatory architecture and how this architecture involves in various forms of transcription network motifs to regulate gene expression.

In male mice, the transcription factors STRA8 and MEISON initiate meiosis I. We report that STRA8/MEISON activates the transcription factors A-MYB and TCFL5, which together reprogram gene expression after spermatogonia enter into meiosis. TCFL5 promotes the transcription of genes required for meiosis, mRNA turnover, miR-34/449 production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5(em1/em1) mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis. Moreover, Tcfl5(+/em1) mutants produce fewer motile sperm.