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Abstract The distal region of the uterine (Fallopian) tube is commonly associated with high-grade serous carcinoma (HGSC), the predominant and most aggressive form of ovarian or extra-uterine cancer. Specific cell states and lineage dynamics of the adult tubal epithelium (TE) remain insufficiently understood, hindering efforts to determine the cell of origin for HGSC. Here, we report a comprehensive census of cell types and states of the mouse uterine tube. We show that distal TE cells expressing the stem/progenitor cell marker Slc1a3 can differentiate into both secretory (Ovgp1+) and ciliated (Fam183b+) cells. Inactivation of Trp53 and Rb1, whose pathways are commonly altered in HGSC, leads to elimination of targeted Slc1a3+ cells by apoptosis, thereby preventing their malignant transformation. In contrast, pre-ciliated cells (Krt5+, Prom1+, Trp73+) remain cancer-prone and give rise to serous tubal intraepithelial carcinomas and overt HGSC. These findings identify transitional pre-ciliated cells as a cancer-prone cell state and point to pre-ciliation mechanisms as diagnostic and therapeutic targets.

Abstract Background MicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. Results We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. Conclusion The present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.

The molecular mechanisms underlying deficits in Down syndrome remain unclear. Here, the authors show that copy number restoration of a chromatin remodeler in trisomic mice is sufficient to rescue epigenomic, physiological and cognitive deficits.

Proper meiotic chromosome segregation requires mismatch repair genes MLH1 and MLH3, of which variants occur in the human population. Here, the authors use computational predictions and yeast assays to select human MLH1/3 variants for modelling in mice, observing reproductive defects from abnormal levels of crossing over.

Abstract Background Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Results Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. Conclusions PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

Cancers arising from the gastric squamous-columnar junction have high incidence and are characterized by a poor prognosis. Here, the authors use genetic mouse models to show that loss of p53 and Rb1 expression results in preferential tumour development at the gastric squamous-columnar junction that contains a large pool of osteopontin responsive Lgr5-CD44+ cells.

Low frequency coding single-nucleotide variants (SNVs) are predicted to disproportionately affect protein function. Here, the authors evaluate 2,009 missense SNVs across 2,185 protein-protein interactions using yeast two-hybrid and protein complementation assays and find that disruptive SNVs often occur in disease-associated genes.

Telomere-led rapid prophase movements (RPMs) in meiotic prophase have been observed in diverse eukaryote species. A shared feature of RPMs is that the force that drives the chromosomal movements is transmitted from the cytoskeleton, through the nuclear envelope, to the telomeres. Studies in mice suggested that dynein movement along microtubules is transmitted to telomeres through SUN1/KASH5 nuclear envelope bridges to generate RPMs. We monitored RPMs in mouse seminiferous tubules using 4D fluorescence imaging and quantitative motion analysis to characterize patterns of movement in the RPM process. We find that RPMs reflect a combination of nuclear rotation and individual chromosome movements. The telomeres move along microtubule tracks that are apparently continuous with the cytoskeletal network and exhibit characteristic arrangements at different stages of prophase. Quantitative measurements confirmed that SUN1/KASH5, microtubules, and dynein, but not actin, were necessary for RPMs and that defects in meiotic recombination and synapsis resulted in altered RPMs.

The miR-34 family was originally found to be a direct target of p53 and is a group of putative tumor suppressors. Surprisingly, mice lacking all mir-34 genes show no increase in cancer formation by 18 months of age, hence placing the physiological relevance of previous studies in doubt. Here, we report that mice with prostate epithelium-specific inactivation of mir-34 and p53 show expansion of the prostate stem cell compartment and develop early invasive adenocarcinomas and high-grade prostatic intraepithelial neoplasia, whereas no such lesions are observed after inactivation of either the mir-34 or p53 genes alone by 15 months of age. Consistently, combined deficiency of p53 and miR-34 leads to acceleration of MET-dependent growth, self-renewal, and motility of prostate stem/progenitor cells. Our study provides direct genetic evidence that mir-34 genes are bona fide tumor suppressors and identifies joint control of MET expression by p53 and miR-34 as a key component of prostate stem cell compartment regulation, aberrations in which may lead to cancer.

Abstract Assisted reproduction procedures often encounter an issue called oocyte maturation arrest (OMA), which is manifested as failed IVF/ICSI attempts using oocytes from some infertile women. In this issue of EMBO Molecular Medicine, Wang et al identify infertile women bearing novel DNA sequence variants in a gene called PABPC1L, which is essential for translation of maternal mRNAs. By conducting a series of in vitro and in vivo experiments, they demonstrated certain variants as being causal for OMA, confirming a conserved requirement for PABPC1L in human oocyte maturation. This study offers a promising therapeutic target for treating OMA patients.

Genetic diversity in offspring is induced by meiotic recombination, which is initiated between homologs at >200 sites originating from meiotic double-strand breaks (DSBs). Of this initial pool, only 1-2 DSBs per homolog pair will be designated to form meiotic crossovers (COs), where reciprocal genetic exchange occurs between parental chromosomes. Cyclin-dependent kinase 2 (CDK2) is known to localize to so-called "late recombination nodules" (LRNs) marking incipient CO sites. However, the role of CDK2 kinase activity in the process of CO formation remains uncertain. Here, we describe the phenotype of 2 Cdk2 point mutants with elevated or decreased activity, respectively. Elevated CDK2 activity was associated with increased numbers of LRN-associated proteins, including CDK2 itself and the MutL homolog 1 (MLH1) component of the MutLγ complex, but did not lead to increased numbers of COs. In contrast, reduced CDK2 activity leads to the complete absence of CO formation during meiotic prophase I. Our data suggest an important role for CDK2 in regulating MLH1 focus numbers and that the activity of this kinase is a key regulatory factor in the formation of meiotic COs.

Circumstances that compromise efficient DNA replication, such as disruptions to replication fork progression, cause a state known as DNA replication stress (RS). Whereas normally proliferating cells experience low levels of RS, excessive RS from intrinsic or extrinsic sources can trigger cell cycle arrest and senescence. Here, we report that a key driver of RS-induced senescence is active downregulation of the Minichromosome Maintenance 2-7 (MCM2-7) factors that are essential for replication origin licensing and which constitute the replicative helicase core. Proliferating cells produce high levels of MCM2-7 that enable formation of dormant origins that can be activated in response to acute, experimentally-induced RS. However, little is known about how physiological RS levels impact MCM2-7 regulation. We found that chronic exposure of primary mouse embryonic fibroblasts (MEFs) to either genetically-encoded or environmentally-induced RS triggered gradual MCM2-7 repression, followed by inhibition of replication and senescence that could be accelerated by MCM hemizygosity. The MCM2-7 reduction in response to RS is TRP53-dependent, and involves a group of Trp53-dependent miRNAs, including the miR-34 family, that repress MCM expression in replication-stressed cells before they undergo terminal cell cycle arrest. miR-34 ablation partially rescued MCM2-7 downregulation and genomic instability in mice with endogenous RS. Together, these data demonstrate that active MCM2-7 repression is a physiologically important mechanism for RS-induced cell cycle arrest and genome maintenance on an organismal level.

Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.

Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin) machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation.

Integrated analyses of functional genomics data have enormous potential for identifying phenotype-associated genes. Tissue-specificity is an important aspect of many genetic diseases, reflecting the potentially different roles of proteins and pathways in diverse cell lineages. Accounting for tissue specificity in global integration of functional genomics data is challenging, as "functionality" and "functional relationships" are often not resolved for specific tissue types. We address this challenge by generating tissue-specific functional networks, which can effectively represent the diversity of protein function for more accurate identification of phenotype-associated genes in the laboratory mouse. Specifically, we created 107 tissue-specific functional relationship networks through integration of genomic data utilizing knowledge of tissue-specific gene expression patterns. Cross-network comparison revealed significantly changed genes enriched for functions related to specific tissue development. We then utilized these tissue-specific networks to predict genes associated with different phenotypes. Our results demonstrate that prediction performance is significantly improved through using the tissue-specific networks as compared to the global functional network. We used a testis-specific functional relationship network to predict genes associated with male fertility and spermatogenesis phenotypes, and experimentally confirmed one top prediction, Mbyl1. We then focused on a less-common genetic disease, ataxia, and identified candidates uniquely predicted by the cerebellum network, which are supported by both literature and experimental evidence. Our systems-level, tissue-specific scheme advances over traditional global integration and analyses and establishes a prototype to address the tissue-specific effects of genetic perturbations, diseases and drugs.

The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1) participates in the arrest of meiosis in F(1) males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6) from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9) encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6)F(1). To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6)F(1) hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies) not involving Prdm9(B6) also acts in the (PWD×B6)F(1) hybrids, since the correction of hybrid sterility by Prdm9(B6) deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWD)F(1) males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9)F(1), harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F(1) hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9(B6). Therefore, the Prdm9(B6) allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s) absent in the parental strains.

Mutations causing replication stress can lead to genomic instability (GIN). In vitro studies have shown that drastic depletion of the MCM2-7 DNA replication licensing factors, which form the replicative helicase, can cause GIN and cell proliferation defects that are exacerbated under conditions of replication stress. To explore the effects of incrementally attenuated replication licensing in whole animals, we generated and analyzed the phenotypes of mice that were hemizygous for Mcm2, 3, 4, 6, and 7 null alleles, combinations thereof, and also in conjunction with the hypomorphic Mcm4(Chaos3) cancer susceptibility allele. Mcm4(Chaos3/Chaos3) embryonic fibroblasts have ∼40% reduction in all MCM proteins, coincident with reduced Mcm2-7 mRNA. Further genetic reductions of Mcm2, 6, or 7 in this background caused various phenotypes including synthetic lethality, growth retardation, decreased cellular proliferation, GIN, and early onset cancer. Remarkably, heterozygosity for Mcm3 rescued many of these defects. Consistent with a role in MCM nuclear export possessed by the yeast Mcm3 ortholog, the phenotypic rescues correlated with increased chromatin-bound MCMs, and also higher levels of nuclear MCM2 during S phase. The genetic, molecular and phenotypic data demonstrate that relatively minor quantitative alterations of MCM expression, homeostasis or subcellular distribution can have diverse and serious consequences upon development and confer cancer susceptibility. The results support the notion that the normally high levels of MCMs in cells are needed not only for activating the basal set of replication origins, but also "backup" origins that are recruited in times of replication stress to ensure complete replication of the genome.

Cancer cells have acquired mutations that alter their growth. Aneuploidy that typify cancer cells are often assumed to contribute to the abnormal growth characteristics. Here we test the idea of a link between aneuploidy and mutations allowing improved growth, using Saccharomyces cerevisiae containing a mcm4 helicase allele that was shown to cause cancer in mice. Yeast bearing this mcm4 allele are prone to undergoing a "hypermutable phase" characterized by a changing karyotype, ultimately yielding progeny with improved growth properties. When such progeny are returned to a normal karyotype by mating, their improved growth remains. Genetic analysis shows their improved growth is due to mutations in just a few loci. In sum, the effects of the mcm4 allele in mice are recapitulated in yeast, and the aneuploidy is not required to maintain improved growth.

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5' splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I.

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1(Mei11), encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1(Mei11) exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.