Unravelling the complex mechanisms of transgenerational epigenetic inheritance.

There are numerous benefits to elucidating how our environment affects our health: from a greater understanding of adaptation to disease prevention. Evidence shows that stressors we are exposed to during our lifetime might cause disease in our descendants. Transgenerational epigenetic inheritance involves the transmission of 'information' over multiple generations via the gametes independent of the DNA base sequence. Despite extensive research, the epigenetic mechanisms remain unclear. Analysis of model organisms exposed to environmental insults (e.g., diet manipulation, stress, toxin exposure) or carrying mutations in the epigenetic regulatory machinery indicates that inheritance of altered DNA methylation, histone modifications, or non-coding RNAs are key mechanisms. Tracking inherited epigenetic information and its effects for multiple generations is a significant challenge to overcome.G.E.T.B. is supported by a studentship from the Wellcome Trust 4-year PhD programme in Developmental Mechanisms. E.D.W. is a Lister Research Prize fellow and was supported by an Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge joint research grant.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.cbpa.2016.06.00

Transferring Fragments of Paternal Metabolism to the Offspring.

Paternal diet influences offspring metabolism, yet the underlying epigenetic mechanisms are unclear. Recently, Chen et al. (2016) and Sharma et al. (2016) identified tRNA fragments in sperm and the male reproductive tract as possible inherited factors altered by paternal diet that lead to gene misexpression and altered metabolism in offspring.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.cmet.2016.02.01

One-carbon metabolism is required for epigenetic stability in the mouse placenta

One-carbon metabolism, including the folate cycle, has a crucial role in fetal development though its molecular function is complex and unclear. The hypomorphic Mtrrgt allele is known to disrupt one-carbon metabolism, and thus methyl group availability, leading to several developmental phenotypes (e.g., neural tube closure defects, fetal growth anomalies). Remarkably, previous studies showed that some of the phenotypes were transgenerationally inherited. Here, we explored the genome-wide epigenetic impact of one-carbon metabolism in placentas associated with fetal growth phenotypes and determined whether specific DNA methylation changes were inherited. Firstly, methylome analysis of Mtrrgt/gt homozygous placentas revealed genome-wide epigenetic instability. Several differentially methylated regions (DMRs) were identified including at the Cxcl1 gene promoter and at the En2 gene locus, which may have phenotypic implications. Importantly, we discovered hypomethylation and ectopic expression of a subset of ERV elements throughout the genome of Mtrrgt/gt placentas with broad implications for genomic stability. Next, we determined that known spermatozoan DMRs in Mtrrgt/gt males were reprogrammed in the placenta with little evidence of direct or transgenerational germline DMR inheritance. However, some spermatozoan DMRs were associated with placental gene misexpression despite normalisation of DNA methylation, suggesting the inheritance of an alternative epigenetic mechanism. Integration of published wildtype histone ChIP-seq datasets with Mtrrgt/gt spermatozoan methylome and placental transcriptome datasets point towards H3K4me3 deposition at key loci. These data suggest that histone modifications might play a role in epigenetic inheritance in this context. Overall, this study sheds light on the mechanistic complexities of one-carbon metabolism in development and epigenetic inheritance

Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction.

We recently reported the first evidence of placental endoplasmic reticulum (ER) stress in the pathophysiology of human intrauterine growth restriction. Here, we used a mouse model to investigate potential underlying mechanisms. Eif2s1(tm1RjK) mice, in which Ser51 of eukaryotic initiation factor 2 subunit alpha (eIF2α) is mutated, display a 30% increase in basal translation. In Eif2s1(tm1RjK) placentas, we observed increased ER stress and anomalous accumulation of glycoproteins in the endocrine junctional zone (Jz), but not in the labyrinthine zone where physiological exchange occurs. Placental and fetal weights were reduced by 15% (97 mg to 82 mg, p < 0.001) and 20% (1009 mg to 798 mg, p < 0.001), respectively. To investigate whether ER stress affects bioactivity of secreted proteins, mouse embryonic fibroblasts (MEFs) were derived from Eif2s1(tm1RjK) mutants. These MEFs exhibited ER stress, grew 50% slower, and showed reduced Akt-mTOR signalling compared to wild-type cells. Conditioned medium (CM) derived from Eif2s1(tm1RjK) MEFs failed to maintain trophoblast stem cells in a progenitor state, but the effect could be rescued by exogenous application of FGF4 and heparin. In addition, ER stress promoted accumulation of pro-Igf2 with altered glycosylation in the CM without affecting cellular levels, indicating that the protein failed to be processed after release. Igf2 is the major growth factor for placental development; indeed, activity in the Pdk1-Akt-mTOR pathways was decreased in Eif2s1(tm1RjK) placentas, indicating loss of Igf2 signalling. Furthermore, we observed premature differentiation of trophoblast progenitors at E9.5 in mutant placentas, consistent with the in vitro results and with the disproportionate development of the labyrinth and Jz seen in placentas at E18.5. Similar disproportion has been reported in the Igf2-null mouse. These results demonstrate that ER stress adversely affects placental development, and that modulation of post-translational processing, and hence bioactivity, of secreted growth factors contributes to this effect. Placental dysmorphogenesis potentially affects fetal growth through reduced exchange capacity

Mtrr hypomorphic mutation alters liver morphology, metabolism and fuel storage in mice.

Nonalcoholic fatty liver disease (NAFLD) is associated with dietary folate deficiency and mutations in genes required for one‑carbon metabolism. However, the mechanism through which this occurs is unclear. To improve our understanding of this link, we investigated liver morphology, metabolism and fuel storage in adult mice with a hypomorphic mutation in the gene methionine synthase reductase (Mtrrgt ). MTRR enzyme is a key regulator of the methionine and folate cycles. The Mtrrgt mutation in mice was previously shown to disrupt one‑carbon metabolism and cause a wide-spectrum of developmental phenotypes and late adult-onset macrocytic anaemia. Here, we showed that livers of Mtrrgt/gt female mice were enlarged compared to control C57Bl/6J livers. Histological analysis of these livers revealed eosinophilic hepatocytes with decreased glycogen content, which was associated with down-regulation of genes involved in glycogen synthesis (e.g., Ugp2 and Gsk3a genes). While female Mtrrgt/gt livers showed evidence of reduced β-oxidation of fatty acids, there were no other associated changes in the lipidome in female or male Mtrrgt/gt livers compared with controls. Defects in glycogen storage and lipid metabolism often associate with disruption of mitochondrial electron transfer system activity. However, defects in mitochondrial function were not detected in Mtrrgt/gt livers as determined by high-resolution respirometry analysis. Overall, we demonstrated that adult Mtrrgt/gt female mice showed abnormal liver morphology that differed from the NAFLD phenotype and that was accompanied by subtle changes in their hepatic metabolism and fuel storage

Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker.

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance

Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations.

Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrr gt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrr gt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrr gt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrr gt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrr gt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrr gt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrr gt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr +/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrr gt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.Lister Institute of Preventative Medicine Newton International Fellowship, Royal Society AG Leventis Fun

Mutation in Folate Metabolism Causes Epigenetic Instability and Transgenerational Effects on Development

SummaryThe importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters’ uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.PaperFlic

Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker

Funder: Center for Trophoblast ResearchAbstract: The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance

Blastocyst transfer in mice alters the placental transcriptome and growth.

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.This work was supported by grants from the Centre for Trophoblast Research (CTR) (to EDW), Lister Institute for Preventative Medicine (to EDW), and Canadian Institutes for Health Research (to JCC). KM was funded by a Newnham College (Cambridge) studentship and A.G. Leventis scholarship. SJT was funded by a Next Generation Fellowship (CTR). GETB was funded by a Wellcome Trust PhD studentship in Developmental Mechanisms. RSH and MP were funded by the CTR