Role of Tet1 in erasure of genomic imprinting.

Genomic imprinting is an allele-specific gene expression system that is important for mammalian development and function. The molecular basis of genomic imprinting is allele-specific DNA methylation. Although it is well known that the de novo DNA methyltransferases Dnmt3a and Dnmt3b are responsible for the establishment of genomic imprinting, how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains unclear. Tet1 is one of the ten-eleven translocation family proteins, which have the capacity to oxidize 5-methylcytosine (5mC), specifically expressed in reprogramming PGCs. Here we report that Tet1 has a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1 knockout males and wild-Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal loss of Tet1 function. Genome-wide DNA methylation analysis of embryonic day 13.5 PGCs and sperm of Tet1 knockout mice revealed hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Analysis of the DNA methylation dynamics in reprogramming PGCs indicates that Tet1 functions to wipe out remaining methylation, including imprinted genes, at the late reprogramming stage. Furthermore, we provide evidence supporting the role of Tet1 in the erasure of paternal imprints in the female germ line. Thus, our study establishes a critical function of Tet1 in the erasure of genomic imprinting.

The Chromatin Regulator BRPF3 Preferentially Activates the HBO1 Acetyltransferase but Is Dispensable for Mouse Development and Survival.

To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and plant homeodomain-linked finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two plant homeodomain-linked fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here, we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (also known as KAT7) and two other subunits but not with related acetyltransferases such as MOZ, MORF, TIP60, and MOF (also known as KAT6A, KAT6B, KAT5, and KAT8, respectively). We have also characterized a mutant mouse strain with a lacZ reporter inserted at the Brpf3 locus. Systematic analysis of ß-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-quantitative PCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2.

Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual FAAH Activity in an LPS-Induced Embryo Resorption Model.

Maternal infections with gram-negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)-activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue plays an important role in the establishment of a local inflammatory process that results in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS-induced deleterious effects during early pregnancy loss. Here, we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved. PBMC isolated from 7-day pregnant mice subjected to different treatments were co-cultured in a transwell system with decidual tissue from control 7-day pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measured by radioconvertion, total decidual protein nitration by Western blot (WB), and decidual FAAH nitration by immunoprecipitation followed by WB. We found that co-culture of PBMC obtained from LPS-treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co-treatment with progesterone or aminoguanidine prevented this effect. We found that LPS-treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. J. Cell. Physiol. 232: 1441-1447, 2017. © 2016 Wiley Periodicals, Inc.

A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog.

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells. Ablation of Hdac1 or Hdac2 by Nestin-Cre had no obvious consequences on brain development and architecture owing to compensation by the paralog. By contrast, combined deletion of Hdac1 and Hdac2 resulted in impaired chromatin structure, DNA damage, apoptosis and embryonic lethality. To dissect the individual roles of HDAC1 and HDAC2, we expressed single alleles of either Hdac1 or Hdac2 in the absence of the respective paralog in neural cells. The DNA-damage phenotype observed in double knockout brains was prevented by expression of a single allele of either Hdac1 or Hdac2. Strikingly, Hdac1(-/-)Hdac2(+/-) brains showed normal development and no obvious phenotype, whereas Hdac1(+/-)Hdac2(-/-) mice displayed impaired brain development and perinatal lethality. Hdac1(+/-)Hdac2(-/-) neural precursor cells showed reduced proliferation and premature differentiation mediated by overexpression of protein kinase C, delta, which is a direct target of HDAC2. Importantly, chemical inhibition or knockdown of protein kinase C delta was sufficient to rescue the phenotype of neural progenitor cells in vitro. Our data indicate that HDAC1 and HDAC2 have a common function in maintaining proper chromatin structures and show that HDAC2 has a unique role by controlling the fate of neural progenitors during normal brain development.

RIP3 mediates the embryonic lethality of caspase-8-deficient mice.

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

Mouse knock-out of IOP1 protein reveals its essential role in mammalian cytosolic iron-sulfur protein biogenesis.

Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.

The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice.

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P(2) and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve(KO/KO) mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve(flox/flox) embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve(WT/KO) mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve(WT/KO) mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50-55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P(2) and PtdIns5P selectively decreased, but this reduction (35-40%) was 10-15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve(WT/KO) mice is able to support the demands for PtdIns(3,5)P(2)/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.

Lipopolysaccharide-induced murine embryonic resorption involves nitric oxide-mediated inhibition of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

The initial inactivation of prostaglandins (PGs) is mediated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). PGs are potent mediators of several biological processes, including inflammation and reproduction. In uterus, PGs play a key role in infection-induced pregnancy loss, in which concentration of this mediator increased. This process is accompanied with the induction of nitric oxide synthase expression and a marked increase in uterine levels of nitric oxide. There is no information concerning nitric oxide contribution to potential changes in PG catabolism, but experimental evidence suggests that nitric oxide modulates PG pathways. The specific objectives of the study were to evaluate the protein expression of HPGD (15-PGDH) and to characterize the nitric oxide-dependent regulation of this enzyme in a model of lipopolysaccharide (LPS)-induced embryonic resorption. Results show that LPS decreased HPGD protein expression and augmented PGE synthase activity; therefore, PGE2 levels increased in uterus in this inflammatory condition. Just as LPS, the treatment with a nitric oxide donor diminished HPGD protein expression in uterine tissue. In contrast, the inhibition of nitric oxide synthesis both in control and in LPS-treated mice increased 15-PGDH levels. Also, we have found that this enzyme and PGE2 levels are not modulated by peroxynitrite, an oxidant agent derived from nitric oxide. This study suggests that LPS and nitric oxide promote a decrease in the ability of the uterus for PG catabolism during bacterially triggered pregnancy loss in mice.

Embryos generated from oocytes lacking complex N- and O-glycans have compromised development and implantation.

Female mice generating oocytes lacking complex N- and O-glycans (double mutants (DM)) produce only one small litter before undergoing premature ovarian failure (POF) by 3 months. Here we investigate the basis of the small litter by evaluating ovulation rate and embryo development in DM (Mgat1(F/F)C1galt1(F/F):ZP3Cre) and Control (Mgat1(F/F)C1galt1(F/F)) females. Surprisingly, DM ovulation rate was normal at 6 weeks, but declined dramatically by 9 weeks. In vitro development of zygotes to blastocysts was equivalent to Controls although all embryos from DM females lacked a normal zona pellucida (ZP) and ∼30% lacked a ZP entirely. In contrast, in vivo preimplantation development resulted in less embryos recovered from DM females compared with Controls at 3.5 days post coitum (dpc) (3.2±1.3 vs 7.0±0.6). Furthermore, only 45% of mated DM females contained embryos at 3.5 dpc. Of the preimplantation embryos collected from DM females, approximately half were morulae unlike Controls where the majority were blastocysts, indicating delayed embryo development in DM females. Post-implantation development in DM females was analysed to determine whether delayed preimplantation development affected subsequent development. In DM females at 5.5 dpc, only ∼40% of embryos found at 3.5 dpc had implanted. However, at 6.5 dpc, implantation sites in DM females corresponded to embryo numbers at 3.5 dpc indicating delayed implantation. At 9.5 dpc, the number of decidua corresponded to embryo numbers 6 days earlier indicating that all implanted embryos progress to midgestation. Therefore, a lack of complex N- and O-glycans in oocytes during development impairs early embryo development and viability in vivo leading to delayed implantation and a small litter.

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation.

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.

Regulation of hematopoiesis by the K63-specific ubiquitin-conjugating enzyme Ubc13.

The ubiquitin-conjugating enzyme Ubc13 mediates lysine-63-specific protein ubiquitination involved in signal transduction by immune receptors; however, the in vivo physiological functions of Ubc13 remain incompletely understood. Using Ubc13 conditional knockout mice, we show that somatic deletion of the Ubc13 gene causes severe loss of multi lineages of immune cells, which is associated with profound atrophy of the thymus and bone marrow, as well as lethality of the mice. Ubc13 has a cell-intrinsic function in mediating hematopoiesis and is essential for the survival and accumulation of hematopoietic stem cells in the bone marrow. Interestingly, loss of Ubc13 results in accumulation of beta-catenin and hyperexpression of Wnt target genes, a condition known to cause impaired hematopoiesis. These results establish Ubc13 as a crucial regulator of hematopoiesis and suggest a role for Ubc13 in the control of Wnt signaling in hematopoietic stem cells.

Indispensable function for embryogenesis, expression and regulation of the nonspecific form of the 5-aminolevulinate synthase gene in mouse.

The first step of heme biosynthesis in animals is catalyzed by 5-aminolevulinate synthase (ALAS), which controls heme supply in various tissues. To clarify the roles that the nonspecific isoform of ALAS (ALAS-N) plays in vivo, we prepared a green fluorescent protein (GFP) knock-in mouse line in which the Alas1 gene (encoding ALAS-N) is replaced with a gfp gene. We found that mice bearing a homozygous knock-in allele (Alas1(GFP/GFP)) were lethal by embryonic day 8.5, demonstrating that ALAS-N is essential for early embryogenesis. Fluorescence microscopic and flow cytometric analyses of heterozygous mouse (Alas1(+/GFP)) tissues showed that the Alas1 expression level differs substantially in tissues; Alas1 is highly expressed in testis Leydig cells, exocrine glands (including submandibular and parotid glands), endocrine glands (such as adrenal and thyroid glands) and hematopoietic lineage cells (including neutrophils and eosinophils). Quantitative analyses of GFP mRNA and ALAS-N mRNA in various tissues of Alas1(+/GFP) mice suggested that the destabilization of ALAS-N mRNA was not uniform in the various tissues. These results thus lay bare that elaborate control of the endogenous heme supply operates in various mouse tissues through regulation of the ALAS-N expression level and that this control is essential for heme homeostasis in animals.

BCKDK regulates the TCA cycle through PDC in the absence of PDK family during embryonic development.

Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that PDK family is dispensable for murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios but died by postnatal day 4 because of hypoglycemia or ketoacidosis. Moreover, PDC was phosphorylated in these embryos, suggesting that another kinase compensates for PDK family. Bioinformatic analysis implicated branched-chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched-chain amino acids (BCAAs) catabolism. Indeed, knockout of Bckdk and Pdk family led to the loss of PDC phosphorylation, an increase in PDC activity and pyruvate entry into the TCA cycle, and embryonic lethality. These findings reveal a regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.

Immune status of decidual macrophages is dependent on the CCL2/CCR2/JAK2 pathway during early pregnancy.

PROBLEM: Decidual macrophages (dMφ ) play an important role in the formation of maternal-fetal immune tolerance. However, factors that influence the immune status of dMφ and the related potential mechanisms have not been elucidated to date. METHOD OF STUDY: The gene transcription in dMφ , decidual stromal cells (DSCs), extravillous trophoblasts (EVTs), and peripheral monocytes (pMo) from human samples were measured using real-time polymerase chain reaction (PCR). Monocyte-DSC co-culture was established to explore whether DSCs influenced dMφ polarization via C-C motif ligand 2 (CCL2)-C-C chemokine receptor (CCR2) binding using flow cytometry. In vivo, changes in dMφ percentage and M1 and M2 marker expression after treatment with CCR2 or Janus kinase 2 (JAK2) inhibitor were detected with flow cytometry. Embryo resorption percentages in the above groups were also analyzed. RESULTS: We found that dMφ were an M1/M2 mixed status at the maternal-fetal interface during early pregnancy. CCL2 influenced the immune status of dMφ in an autocrine and paracrine manner. As a downstream regulator of CCR2 and triggers the Stat3 pathway, JAK2 was found to be essential for dMφ homeostasis in vivo. JAK2 inhibitor decreased the dMφ proportion and attenuated Ki67, CD36, CD86, CD206, TNF, and IL-10 expression in dMφ at E8.5 d. Moreover, CCR2-JAK2 pathway inhibition decreased the width of the placental labyrinth layer, further influencing the pregnancy outcome. CONCLUSION: The M1/M2 mixed immune status of dMφ was regulated by DSCs via CCR2, and the CCL2/CCR2/JAK2 pathway was essential for the immune status of dMφ and the outcome of early pregnancy.

Generation of a mouse model with a reversible hypomorphic cytochrome P450 reductase gene: utility for tissue-specific rescue of the reductase expression, and insights from a resultant mouse model with global suppression of P450 reductase expression in extrahepatic tissues.

A mouse model termed Cpr-low (CL) was recently generated, in which the expression of the cytochrome P450 reductase (Cpr) gene was globally down-regulated. The decreased CPR expression was accompanied by phenotypical changes, including reduced embryonic survival, decreases in circulating cholesterol, increases in hepatic P450 expression, and female infertility (accompanied by elevated serum testosterone and progesterone levels). In the present study, a complementary mouse model [named reversible-CL (r-CL)] was generated, in which the reduced CPR expression can be reversed in an organ-specific fashion. The neo cassette, which was inserted into the last Cpr intron in r-CL mice, can be deleted by Cre recombinase, thus returning the structure of the Cpr gene (and hence CPR expression) to normal in Cre-expressing cells. All previously identified phenotypes of the CL mice were preserved in the r-CL mice. As a first application of the r-CL model, we have generated an extrahepatic-CL (xh-CL) mouse for testing of the functions of CPR-dependent enzymes in all extrahepatic tissues. The xh-CL mice, generated by mating of r-CL mice with albumin-Cre mice, had normal CPR expression in hepatocytes but down-regulated CPR expression elsewhere. They were indistinguishable from wild-type mice in body and liver weights, circulating cholesterol levels, and hepatic microsomal P450 expression and activities; however, they still showed elevated serum testosterone and progesterone levels and sterility in females. Embryonic lethality was prevented in males, but apparently not in females, indicating a critical role for fetal hepatic CPR-dependent enzymes in embryonic development, at least in males.

Differential involvement of the extracellular 6-O-endosulfatases Sulf1 and Sulf2 in brain development and neuronal and behavioural plasticity.

The extracellular sulfatases Sulf1 and Sulf2 remove specific 6-O-sulfate groups from heparan sulfate, thereby modulating numerous signalling pathways underlying development and homeostasis. In vitro data have suggested that the two enzymes show functional redundancy. To elucidate their in vivo functions and to further address the question of a putative redundancy, we have generated Sulf1- and Sulf2-deficient mice. Phenotypic analysis of these animals revealed higher embryonic lethality of Sulf2 knockout mice, which can be associated with neuroanatomical malformations during embryogenesis. Sulf1 seems not to be essential for developmental or postnatal viability, as mice deficient in this sulfatase show no overt phenotype. However, neurite outgrowth deficits were observed in hippocampal and cerebellar neurons of both mutant mouse lines, suggesting that not only Sulf2 but also Sulf1 function plays a role in the developing nervous system. Behavioural analysis revealed differential deficits with regard to cage activity and spatial learning for Sulf1- and Sulf2-deficient mouse lines. In addition, Sulf1-specific deficits were shown for synaptic plasticity in the CA1 region of the hippocampus, associated with a reduced spine density. These results reveal that Sulf1 and Sulf2 fulfil non-redundant functions in vivo in the development and maintenance of the murine nervous system.

Methylenetetrahydrofolate reductase deficiency and low dietary folate increase embryonic delay and placental abnormalities in mice.

BACKGROUND: Despite extensive research on mild methylenetetrahydrofolate reductase (MTHFR) deficiency and low dietary folate in different disorders, the association of these metabolic disturbances with a variety of congenital defects and pregnancy complications remains controversial. In this study we investigated the effects of MTHFR and dietary folate deficiency at 10.5 days post coitum (dpc) in our mouse model of mild MTHFR deficiency. METHODS: Mthfr +/+ and +/- female mice were fed a control or folic acid-deficient diet for 6 weeks, then mated with Mthfr +/- males. At 10.5 dpc, embryos were examined and placentae were collected for histologic evaluation. RESULTS: Maternal MTHFR and folate deficiencies resulted in increased developmental delays and smaller embryos. We also observed a low frequency of a variety of embryonic defects in the experimental groups, such as neural tube, heart looping, and turning defects; these results mimic the low incidence and multifactorial nature of these anomalies in humans. Folate-deficient mice also had increased embryonic losses and severe placental defects, including placental abruption and disturbed patterning of placental layers. Folate-deficient placentae had decreased ApoA-I expression, and there was a trend toward a negative correlation between ApoA-I expression with maternal homocysteine concentrations. CONCLUSIONS: Our study provides biological evidence linking maternal MTHFR and dietary folate deficiencies to adverse pregnancy outcomes in mice. It underscores the importance of folate not only in reducing the incidence of early embryonic defects, but also in the prevention of developmental delays and placental abnormalities that may increase susceptibility to other defects and to reproductive complications.

The leukodystrophy mutation Polr3b R103H causes homozygote mouse embryonic lethality and impairs RNA polymerase III biogenesis.

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3aG672E/G672E/Polr3b+/R103Hdouble mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.

Premature activation of Cdk1 leads to mitotic events in S phase and embryonic lethality.

Cell cycle regulation, especially faithful DNA replication and mitosis, are crucial to maintain genome stability. Cyclin-dependent kinase (CDK)/cyclin complexes drive most processes in cellular proliferation. In response to DNA damage, cell cycle surveillance mechanisms enable normal cells to arrest and undergo repair processes. Perturbations in genomic stability can lead to tumor development and suggest that cell cycle regulators could be effective targets in anticancer therapy. However, many clinical trials ended in failure due to off-target effects of the inhibitors used. Here, we investigate in vivo the importance of WEE1- and MYT1-dependent inhibitory phosphorylation of mammalian CDK1. We generated Cdk1AF knockin mice, in which two inhibitory phosphorylation sites are replaced by the non-phosphorylatable amino acids T14A/Y15F. We uncovered that monoallelic expression of CDK1AF is early embryonic lethal in mice and induces S phase arrest accompanied by γH2AX and DNA damage checkpoint activation in mouse embryonic fibroblasts (MEFs). The chromosomal fragmentation in Cdk1AF MEFs does not rely on CDK2 and is partly caused by premature activation of MUS81-SLX4 structure-specific endonuclease complexes, as well as untimely onset of chromosome condensation followed by nuclear lamina disassembly. We provide evidence that tumor development in liver expressing CDK1AF is inhibited. Interestingly, the regulatory mechanisms that impede cell proliferation in CDK1AF expressing cells differ partially from the actions of the WEE1 inhibitor, MK-1775, with p53 expression determining the sensitivity of cells to the drug response. Thus, our work highlights the importance of improved therapeutic strategies for patients with various cancer types and may explain why some patients respond better to WEE1 inhibitors.