Publications by authors named "John C Schimenti"

106 Publications

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

Genome Biol 2021 Mar 16;22(1):83. Epub 2021 Mar 16.

Department of Medicine, Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, 30912, USA.

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.
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http://dx.doi.org/10.1186/s13059-021-02304-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962346PMC
March 2021

Strategies to Identify Genetic Variants Causing Infertility.

Trends Mol Med 2021 Jan 8. Epub 2021 Jan 8.

Cornell University, College of Veterinary Medicine, Department of Biomedical Sciences, Ithaca, NY 14853, USA. Electronic address:

Genetic causes are thought to underlie about half of infertility cases, but understanding the genetic bases has been a major challenge. Modern genomics tools allow more sophisticated exploration of genetic causes of infertility through population, family-based, and individual studies. Nevertheless, potential therapies based on genetic diagnostics will be limited until there is certainty regarding the causality of genetic variants identified in an individual. Genome modulation and editing technologies have revolutionized our ability to functionally test such variants, and also provide a potential means for clinical correction of infertility variants. This review addresses strategies being used to identify causative variants of infertility.
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http://dx.doi.org/10.1016/j.molmed.2020.12.008DOI Listing
January 2021

Sexually dimorphic DNA damage responses and mutation avoidance in the mouse germline.

Genes Dev 2020 Dec 12;34(23-24):1637-1649. Epub 2020 Nov 12.

Department of Biomedical Sciences,, Cornell University, Ithaca, New York 14853, USA.

Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared with somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon derepression. We also used whole-genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells ( ) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.
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http://dx.doi.org/10.1101/gad.341602.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706705PMC
December 2020

Variants in RABL2A causing male infertility and ciliopathy.

Hum Mol Genet 2020 Dec;29(20):3402-3411

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.
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http://dx.doi.org/10.1093/hmg/ddaa230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749704PMC
December 2020

A novel function for CDK2 activity at meiotic crossover sites.

PLoS Biol 2020 10 19;18(10):e3000903. Epub 2020 Oct 19.

Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology, and Research), Singapore, Republic of Singapore.

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.
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http://dx.doi.org/10.1371/journal.pbio.3000903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595640PMC
October 2020

Conditional surrender in one generation: determining the reproductive roles of mouse embryo lethal genes by embryo complementation.

Authors:
John C Schimenti

Biol Reprod 2021 Jan;104(1):8-10

Dept. of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA.

The laboratory mouse is the most widely used animal model for studying the genetics and biology of mammalian development and reproduction. Embryonic stem cell (ESC) gene targeting technology, and the sophisticated genomic manipulations it allowed, was unique to this organism for a long period of time; this was a major factor in the mouse's rise to pre-eminence as a model system over the past three decades or so. The recent advent of CRISPR/Cas9 technology has democratized the application of genome editing to essentially all organisms. Nevertheless, the scientific infrastructure behind the mouse still makes it the organism of choice for studying molecular mechanisms of mammalian development, and for modeling human development and disease.
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http://dx.doi.org/10.1093/biolre/ioaa193DOI Listing
January 2021

Most Commonly Mutated Genes in High-Grade Serous Ovarian Carcinoma Are Nonessential for Ovarian Surface Epithelial Stem Cell Transformation.

Cell Rep 2020 09;32(9):108086

Department of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA. Electronic address:

High-grade serous ovarian carcinoma (HGSOC) is the fifth leading cause of cancer-related deaths of women in the United States. Disease-associated mutations have been identified by the Cancer Genome Atlas Research Network. However, aside from mutations in TP53 or the RB1 pathway that are common in HGSOC, the contributions of mutation combinations are unclear. Here, we report CRISPR mutagenesis of 20 putative HGSOC driver genes to identify combinatorial disruptions of genes that transform either ovarian surface epithelium stem cells (OSE-SCs) or non-stem cells (OSE-NSs). Our results support the OSE-SC theory of HGSOC initiation and suggest that most commonly mutated genes in HGSOC have no effect on OSE-SC transformation initiation. Our results indicate that disruption of TP53 and PTEN, combined with RB1 disruption, constitutes a core set of mutations driving efficient transformation in vitro. The combined data may contribute to more accurate modeling of HGSOC development.
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http://dx.doi.org/10.1016/j.celrep.2020.108086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545232PMC
September 2020

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 () gene.

Proc Natl Acad Sci U S A 2020 06 3;117(24):13680-13688. Epub 2020 Jun 3.

Human Developmental Genetics Unit, Institut Pasteur, 75724 Paris, France;

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene is present in many cases, the etiology is unknown in most -negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families ( = 4.4 × 10), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations ( < 1.8 × 10). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.
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http://dx.doi.org/10.1073/pnas.1921676117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306989PMC
June 2020

A reporter mouse for in vivo detection of DNA damage in embryonic germ cells.

Genesis 2020 08 28;58(8):e23368. Epub 2020 Apr 28.

Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1-mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell-specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB-inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose- and time-dependent manner. The dynamic time-sensitive and dose-sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.
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http://dx.doi.org/10.1002/dvg.23368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897368PMC
August 2020

Oocyte Elimination Through DNA Damage Signaling from CHK1/CHK2 to p53 and p63.

Genetics 2020 06 9;215(2):373-378. Epub 2020 Apr 9.

Department of Biomedical Sciences and Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850

Eukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance because of the requirement for SPO11-induced programmed double-strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (, mutants), but also oocytes that are defective in homologous chromosome synapsis and accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both p53 (TRP53) and the oocyte-exclusive isoform of p63, TAp63, protects nearly all and oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semiredundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.
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http://dx.doi.org/10.1534/genetics.120.303182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7268994PMC
June 2020

Meiotic epigenetic factor PRDM9 impacts sperm quality of hybrid mice.

Reproduction 2020 07;160(1):53-64

Laboratory of Germ Cell Development, Division BIOCEV, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague 4, Czech Republic.

Reduced fertility of male mouse hybrids relative to their parents, or hybrid sterility, is governed by the hybrid sterility 1 (Hst1) locus. Rescue experiments with transgenes carrying sequences within or near Hst1 manifested that Hst1 contains the gene encoding meiosis-specific histone methyltransferase PRDM9. The Prdm9 gene is responsible for partial meiotic arrest, testicular atrophy, and low sperm count in (C57BL/6J x PWD)F1 mouse hybrids. Here we report that these male hybrids suffer an additional reproductive disadvantage, decreased sperm quality, which is (i) further exacerbated by the introduction of long transgenes carrying sequences from Hst1 with incomplete Prdm9 into their genome and (ii) controlled by the Prdm9 dosage. These transgenic male hybrids displayed the features of severe oligoasthenoteratozoospermia (OAT), a human infertility syndrome characterized by a low number of spermatozoa with poor motility and morphological abnormalities. Analysis of spermiogenesis in these mice revealed acrosome detachment, aberrant elongation and condensation of the nucleus. As a result, the transgenic sperm had acrosome malformations, abnormal chromatin packaging, and fragmented DNA with elevated base oxidation, revealed by using multiple methods. Heterozygosity for one null Prdm9 allele improved meiotic progression and sperm quality of both non- and transgenic hybrids. Our results indicate that genomic analysis of OAT patients should include consideration of allelic variants in PRDM9, and our transgenic models can serve as tools to understand the diverse molecular processes that, when perturbed, can cause this disease.
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http://dx.doi.org/10.1530/REP-19-0528DOI Listing
July 2020

SKP1 drives the prophase I to metaphase I transition during male meiosis.

Sci Adv 2020 03 25;6(13):eaaz2129. Epub 2020 Mar 25.

Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, USA.

The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.
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http://dx.doi.org/10.1126/sciadv.aaz2129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096161PMC
March 2020

Copy-number variation introduced by long transgenes compromises mouse male fertility independently of pachytene checkpoints.

Chromosoma 2020 03 15;129(1):69-82. Epub 2020 Jan 15.

Laboratory of Germ Cell Development, Division BIOCEV, Institute of Molecular Genetics of the Czech Academy of Sciences, 14220, Prague, Czech Republic.

Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18 days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.
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http://dx.doi.org/10.1007/s00412-019-00730-8DOI Listing
March 2020

Gastric squamous-columnar junction contains a large pool of cancer-prone immature osteopontin responsive Lgr5CD44 cells.

Nat Commun 2020 01 3;11(1):84. Epub 2020 Jan 3.

Department of Biomedical Sciences and Cornell Stem Cell Program, Cornell University, Ithaca, NY, 14853, USA.

Areas of a junction between two types of epithelia are known to be cancer-prone in many organ systems. However, mechanisms for preferential malignant transformation at the junction areas remain insufficiently elucidated. Here we report that inactivation of tumor suppressor genes Trp53 and Rb1 in the gastric squamous-columnar junction (SCJ) epithelium results in preferential formation of metastatic poorly differentiated neoplasms, which are similar to human gastroesophageal carcinoma. Unlike transformation-resistant antral cells, SCJ cells contain a highly proliferative pool of immature Lgr5CD44 cells, which are prone to transformation in organoid assays, comprise early dysplastic lesions, and constitute up to 30% of all neoplastic cells. CD44 ligand osteopontin (OPN) is preferentially expressed in and promotes organoid formation ability and transformation of the SCJ glandular epithelium. OPN and CD44 overexpression correlate with the worst prognosis of human gastroesophageal carcinoma. Thus, detection and selective targeting of the active OPN-CD44 pathway may have direct clinical relevance.
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http://dx.doi.org/10.1038/s41467-019-13847-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941991PMC
January 2020

CDK2 kinase activity is a regulator of male germ cell fate.

Development 2019 11 6;146(21). Epub 2019 Nov 6.

Cornell University, College of Veterinary Medicine, Department of Biomedical Sciences, Ithaca, NY 14853, USA

The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter balancing SSC renewal versus terminal differentiation. Here, we report that precise regulation of the cell cycle is crucial for this balance. Whereas cyclin-dependent kinase 2 () is not necessary for mouse viability or gametogenesis stages prior to meiotic prophase I, mice bearing a deregulated allele ( ) are severely deficient in spermatogonial differentiation. This allele disrupts an inhibitory phosphorylation site (Tyr15) for the kinase WEE1. Remarkably, mice possess abnormal clusters of mitotically active SSC-like cells, but these are eventually removed by apoptosis after failing to differentiate properly. Analyses of lineage markers, germ cell proliferation over time, and single cell RNA-seq data revealed delayed and defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that is a gain-of-function allele causing elevated kinase activity, which underlies these differentiation defects. Our results demonstrate that precise regulation of CDK2 kinase activity in male germ cell development is crucial for the gonocyte-to-spermatogonia transition and long-term spermatogenic homeostasis.
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http://dx.doi.org/10.1242/dev.180273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857589PMC
November 2019

Extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations.

Nat Commun 2019 09 12;10(1):4141. Epub 2019 Sep 12.

Department of Computational Biology, Cornell University, Ithaca, NY, 14853, USA.

Each human genome carries tens of thousands of coding variants. The extent to which this variation is functional and the mechanisms by which they exert their influence remains largely unexplored. To address this gap, we leverage the ExAC database of 60,706 human exomes to investigate experimentally the impact of 2009 missense single nucleotide variants (SNVs) across 2185 protein-protein interactions, generating interaction profiles for 4797 SNV-interaction pairs, of which 421 SNVs segregate at > 1% allele frequency in human populations. We find that interaction-disruptive SNVs are prevalent at both rare and common allele frequencies. Furthermore, these results suggest that 10.5% of missense variants carried per individual are disruptive, a higher proportion than previously reported; this indicates that each individual's genetic makeup may be significantly more complex than expected. Finally, we demonstrate that candidate disease-associated mutations can be identified through shared interaction perturbations between variants of interest and known disease mutations.
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http://dx.doi.org/10.1038/s41467-019-11959-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742646PMC
September 2019

A predicted deleterious allele of the essential meiosis gene MND1, present in ~ 3% of East Asians, does not disrupt reproduction in mice.

Mol Hum Reprod 2019 10;25(10):668-673

Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA.

Infertility is a major health problem affecting ~15% of couples worldwide. Except for cases involving readily detectable chromosome aberrations, confident identification of a causative genetic defect is problematic. Despite the advent of genome sequencing for diagnostic purposes, the preponderance of segregating genetic variants complicates identification of culprit genetic alleles or mutations. Many algorithms have been developed to predict the effects of 'variants of unknown significance', typically single nucleotide polymorphisms (SNPs), but these predictions are not sufficiently accurate for clinical action. As part of a project to identify population variants that impact fertility, we have been generating clustered regularly interspaced short palindromic repeats-Cas9 edited mouse models of suspect SNPs in genes that are known to be required for fertility in mice. Here, we present data on a non-synonymous (amino acid altering) SNP (rs140107488) in the meiosis gene Mnd1, which is predicted bioinformatically to be deleterious to protein function. We report that when modeled in mice, this allele (MND1K85M), which is present at an allele frequency of ~ 3% in East Asians, has no discernable effect upon fertility, fecundity or gametogenesis, although it may cause sex skewing of progeny from homozygous males. In sum, assuming the mouse model accurately reflects the impact of this variant in humans, rs140107488 appears to be a benign allele that can be eliminated or de-prioritized in clinical genomic analyses of infertility patients.
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http://dx.doi.org/10.1093/molehr/gaz048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821341PMC
October 2019

A segregating human allele of SPO11 modeled in mice disrupts timing and amounts of meiotic recombination, causing oligospermia and a decreased ovarian reserve†.

Biol Reprod 2019 08;101(2):347-359

Department of Biomedical Sciences and the Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853.

A major challenge in medical genetics is to characterize variants of unknown significance (VUS). Doing so would help delineate underlying causes of disease and the design of customized treatments. Infertility has presented an especially difficult challenge with respect to not only determining if a given patient has a genetic basis, but also to identify the causative genetic factor(s). Though genome sequencing can identify candidate variants, in silico predictions of causation are not always sufficiently reliable so as to be actionable. Thus, experimental validation is crucial. Here, we describe the phenotype of mice containing a non-synonymous (proline-to-threonine at position 306) change in Spo11, corresponding to human SNP rs185545661. SPO11 is a topoisomerase-like protein that is essential for meiosis because it induces DNA double stranded breaks (DSBs) that stimulate pairing and recombination of homologous chromosomes. Although both male and female Spo11P306T/P306T mice were fertile, they had reduced sperm and oocytes, respectively. Spermatocyte chromosomes exhibited synapsis defects (especially between the X and Y chromosomes), elevated apoptotic cells, persistent markers of DSBs, and most importantly, fewer Type 1 crossovers that causes some chromosomes to have none. Spo11P306T/- mice were sterile and made fewer meiotic DSBs than Spo11+/- animals, suggesting that the Spo11P306T allele is a hypomorph and likely is delayed in making sufficient DSBs in a timely fashion. If the consequences are recapitulated in humans, it would predict phenotypes of premature ovarian failure, reduced sperm counts, and possible increased number of aneuploid gametes. These results emphasize the importance of deep phenotyping in order to accurately assess the impact of VUSs in reproduction genes.
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http://dx.doi.org/10.1093/biolre/ioz089DOI Listing
August 2019

Female-biased embryonic death from inflammation induced by genomic instability.

Nature 2019 03 20;567(7746):105-108. Epub 2019 Feb 20.

Cornell University College of Veterinary Medicine, Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA.

Genomic instability can trigger cellular responses that include checkpoint activation, senescence and inflammation. Although genomic instability has been extensively studied in cell culture and cancer paradigms, little is known about its effect during embryonic development, a period of rapid cellular proliferation. Here we report that mutations in the heterohexameric minichromosome maintenance complex-the DNA replicative helicase comprising MCM2 to MCM7-that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. This bias was not attributable to X chromosome-inactivation defects, differential replication licensing or X versus Y chromosome size, but rather to 'maleness'-XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its anti-inflammatory properties. Ibuprofen, a non-steroidal anti-inflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm gene; similar to MCM mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair. In addition, deficiency in the anti-inflammatory IL10 receptor was synthetically lethal with the Mcm4 helicase mutant. Our experiments indicate that, during development, DNA damage associated with DNA replication induces inflammation that is preferentially lethal to female embryos, because male embryos are protected by high levels of intrinsic testosterone.
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http://dx.doi.org/10.1038/s41586-019-0936-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497049PMC
March 2019

A putative human infertility allele of the meiotic recombinase DMC1 does not affect fertility in mice.

Hum Mol Genet 2018 11;27(22):3911-3918

Department of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY, USA.

Whole-exome or whole-genome sequencing is becoming routine in clinical situations for identifying mutations underlying presumed genetic causes of disease including infertility. While this is a powerful approach for implicating polymorphisms or de novo mutations in genes plausibly related to the phenotype, a greater challenge is to definitively prove causality. This is a crucial requisite for treatment, especially for infertility, in which validation options are limited. In this study, we created a mouse model of a putative infertility allele, DMC1M200V. DMC1 encodes a RecA homolog essential for meiotic recombination and fertility in mice. This allele was originally implicated as being responsible for the sterility of a homozygous African woman, a conclusion supported by subsequent biochemical analyses of the mutant protein and by studies of yeast with the orthologous amino acid change. Here, we found that Dmc1M200V/M200V male and female mice are fully fertile and do not exhibit any gonadal abnormalities. Detailed immunocytological analysis of meiosis revealed no defects suggestive of compromised fertility. This study serves as a cautionary tale for making conclusions about consequences of genetic variants, especially with respect to infertility, and emphasizes the importance of conducting relevant biological assays for making accurate diagnoses in the era of genomic medicine.
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http://dx.doi.org/10.1093/hmg/ddy286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216207PMC
November 2018

Unpackaging the genetics of mammalian fertility: strategies to identify the "reproductive genome".

Biol Reprod 2018 12;99(6):1119-1128

The Jackson Laboratory, Bar Harbor, Maine, USA.

Gene mutations, including different alleles of the same gene, are tremendously useful in deconstructing complex developmental systems, such as reproduction, into component molecular pathways. For this reason, great effort has been devoted in the past three decades to biased (reverse genetic) and unbiased (forward genetic) searches for new genes that impact mammalian reproduction and fertility. These efforts have more recently been complemented with international efforts to systematically mutate all mouse genes and to determine their phenotypes (essentially a hybrid of forward and reverse genetics). Here, we survey the available data on the relative productivity of these approaches in identifying fertility genes, estimate the number of protein-coding genes essential for fertility of males and females, and predict the next major directions in the genetics of reproduction and fertility.
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http://dx.doi.org/10.1093/biolre/ioy133DOI Listing
December 2018

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries.

J Vis Exp 2018 05 2(135). Epub 2018 May 2.

Department of Biomedical Sciences, Cornell University; Department of Molecular Biology and Genetics, Cornell University.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.
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http://dx.doi.org/10.3791/57593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101098PMC
May 2018

The DNA Damage Checkpoint Eliminates Mouse Oocytes with Chromosome Synapsis Failure.

Mol Cell 2017 Sep 24;67(6):1026-1036.e2. Epub 2017 Aug 24.

Cornell University, Departments of Biomedical Sciences and Molecular Biology and Genetics, Ithaca, NY 14850, USA. Electronic address:

Pairing and synapsis of homologous chromosomes during meiosis is crucial for producing genetically normal gametes and is dependent upon repair of SPO11-induced double-strand breaks (DSBs) by homologous recombination. To prevent transmission of genetic defects, diverse organisms have evolved mechanisms to eliminate meiocytes containing unrepaired DSBs or unsynapsed chromosomes. Here we show that the CHK2 (CHEK2)-dependent DNA damage checkpoint culls not only recombination-defective mouse oocytes but also SPO11-deficient oocytes that are severely defective in homolog synapsis. The checkpoint is triggered in oocytes that accumulate a threshold level of spontaneous DSBs (∼10) in late prophase I, the repair of which is inhibited by the presence of HORMAD1/2 on unsynapsed chromosome axes. Furthermore, Hormad2 deletion rescued the fertility of oocytes containing a synapsis-proficient, DSB repair-defective mutation in a gene (Trip13) required for removal of HORMADs from synapsed chromosomes, suggesting that many meiotic DSBs are normally repaired by intersister recombination in mice.
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http://dx.doi.org/10.1016/j.molcel.2017.07.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621520PMC
September 2017

Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.

Genetics 2017 08 2;206(4):1823-1828. Epub 2017 Jun 2.

Department of Biomedical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, New York 14853.

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or -fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-H-benzimidazole-5-carboxamide-hydrate ("CHK2iII") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.
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http://dx.doi.org/10.1534/genetics.117.203455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560790PMC
August 2017

Repair of Meiotic DNA Breaks and Homolog Pairing in Mouse Meiosis Requires a Minichromosome Maintenance (MCM) Paralog.

Genetics 2017 02 16;205(2):529-537. Epub 2016 Dec 16.

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

The mammalian Mcm-domain containing 2 (Mcmdc2) gene encodes a protein of unknown function that is homologous to the minichromosome maintenance family of DNA replication licensing and helicase factors. Drosophila melanogaster contains two separate genes, the Mei-MCMs, which appear to have arisen from a single ancestral Mcmdc2 gene. The Mei-MCMs are involved in promoting meiotic crossovers by blocking the anticrossover activity of BLM helicase, a function presumably performed by MSH4 and MSH5 in metazoans. Here, we report that MCMDC2-deficient mice of both sexes are viable but sterile. Males fail to produce spermatozoa, and formation of primordial follicles is disrupted in females. Histology and immunocytological analyses of mutant testes revealed that meiosis is arrested in prophase I, and is characterized by persistent meiotic double-stranded DNA breaks (DSBs), failure of homologous chromosome synapsis and XY body formation, and an absence of crossing over. These phenotypes resembled those of MSH4/5-deficient meiocytes. The data indicate that MCMDC2 is essential for invasion of homologous sequences by RAD51- and DMC1-coated single-stranded DNA filaments, or stabilization of recombination intermediates following strand invasion, both of which are needed to drive stable homolog pairing and DSB repair via recombination in mice.
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http://dx.doi.org/10.1534/genetics.116.196808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289834PMC
February 2017

The Chromatin Remodeling Component Arid1a Is a Suppressor of Spontaneous Mammary Tumors in Mice.

Genetics 2016 08 8;203(4):1601-11. Epub 2016 Jun 8.

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Human cancer genome studies have identified the SWI/SNF chromatin remodeling complex member ARID1A as one of the most frequently altered genes in several tumor types. Its role as an ovarian tumor suppressor has been supported in compound knockout mice. Here, we provide genetic and functional evidence that Arid1a is a bona fide mammary tumor suppressor, using the Chromosome aberrations occurring spontaneously 3 (Chaos3) mouse model of sporadic breast cancer. About 70% of mammary tumors that formed in these mice contained a spontaneous deletion removing all or part of one Arid1a allele. Restoration of Arid1a expression in a Chaos3 mammary tumor line with low Arid1a levels greatly impaired its ability to form tumors following injection into cleared mammary glands, indicating that ARID1A insufficiency is crucial for maintenance of these Trp53-proficient tumors. Transcriptome analysis of tumor cells before and after reintroduction of Arid1a expression revealed alterations in growth signaling and cell-cycle checkpoint pathways, in particular the activation of the TRP53 pathway. Consistent with the latter, Arid1a reexpression in tumor cells led to increased p21 (Cdkn1a) expression and dramatic accumulation of cells in G2 phase of the cell cycle. These results not only provide in vivo evidence for a tumor suppressive and/or maintenance role in breast cancer, but also indicate a potential opportunity for therapeutic intervention in ARID1A-deficient human breast cancer subtypes that retain one intact copy of the gene and also maintain wild-type TRP53 activity.
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http://dx.doi.org/10.1534/genetics.115.184879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981264PMC
August 2016

L. C. Dunn and Donald Charles on Quantitative Traits in the Mouse.

Authors:
John C Schimenti

Genetics 2016 Mar;202(3):867-8

College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

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http://dx.doi.org/10.1534/genetics.116.187534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788122PMC
March 2016

Transcriptional profiling of cortical versus cancellous bone from mechanically-loaded murine tibiae reveals differential gene expression.

Bone 2016 May 12;86:22-9. Epub 2016 Feb 12.

Sibley School of Mechanical and Aerospace Engineering, Cornell University, 105 Upson Hall, Ithaca, NY 14853, USA; Nancy E and Peter C Meinig School of Biomedical Engineering, Cornell University, 101 Weill Hall, Ithaca, NY 14853, USA; Research Division, Hospital for Special Surgery, 541 East 71st St., New York, NY 10021, USA. Electronic address:

Mechanical loading is an anabolic stimulus that increases bone mass, and thus a promising method to counteract osteoporosis-related bone loss. The mechanism of this anabolism remains unclear, and needs to be established for both cortical and cancellous envelopes individually. We hypothesized that cortical and cancellous bone display different gene expression profiles at baseline and in response to mechanical loading. To test this hypothesis, the left tibiae of 10-week-old female C57Bl/6 mice were subjected to one session of axial tibial compression (9N, 1200cycles, 4Hz triangle waveform) and euthanized 3 and 24h following loading. The right limb served as the contralateral control. We performed RNA-seq on marrow-free metaphyseal samples from the cortical shell and the cancellous core to determine differential gene expression at baseline (control limb) and in response to load. Differential expression was verified with qPCR. Cortical and cancellous bone exhibited distinctly different transcriptional profiles basally and in response to mechanical loading. More genes were differentially expressed with loading at 24h with more genes downregulated at 24h than at 3h in both tissues. Enhanced Wnt signaling dominated the response in cortical bone at 3 and 24h, but in cancellous bone only at 3h. In cancellous bone at 24h many muscle-related genes were downregulated. These findings reveal key differences between cortical and cancellous genetic regulation in response to mechanical loading. Future studies at different time points and multiple loading sessions will add to our knowledge of cortical and cancellous mechanotransduction with the potential to identify new targets for mouse genetic knockout studies and drugs to treat osteoporosis.
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http://dx.doi.org/10.1016/j.bone.2016.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833881PMC
May 2016

Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation.

PLoS Genet 2016 Jan 14;12(1):e1005787. Epub 2016 Jan 14.

Department of Biomedical Sciences, Cornell University, Ithaca, New York, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1005787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713100PMC
January 2016

MCM9 deficiency delays primordial germ cell proliferation independent of the ATM pathway.

Genesis 2015 Nov 14;53(11):678-84. Epub 2015 Oct 14.

Department of Biomedical Sciences, Cornell University, Ithaca, New York.

Maintenance of genome integrity is crucial for the germline, and this is reflected by lower mutation rates in gametes than somatic cells. Germ cells at different stages employ different DNA damage response (DDR) mechanisms. In response to certain DNA repair defects, primordial germ cells (PGCs) either undergo apoptosis or delayed proliferation, although little is known about the underlying mechanisms that govern these outcomes. Here, we report genetic studies of DDR pathways that underlie germ cell depletion in mice mutant for minichromosome maintenance 9 (Mcm9), a gene that plays a role in homologous recombination repair (HRR). Germ cell depletion in these mice is a result of reduced PGC numbers both before and after they arrive in the primitive gonads. This reduction was attributable to reduced proliferation, not apoptosis, and this response was independent of ATM-CHK2-TRP53-P21 signaling. This mechanism of PGC depletion differs from that in Fancm mutants, which also display reduced PGC depletion that is partially orchestrated by the ATM-TRP53-P21 pathway. Germ cell depletion in mice doubly deficient for FANCM and MCM9 was additive, indicating that the damage caused by each mutation triggers different DDR pathways to slow the cell cycle as a means to preserve genomic integrity. genesis 53:678-684, 2015. © 2015 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvg.22901DOI Listing
November 2015