The mammalian LINC complex component SUN1 regulates muscle regeneration by modulating drosha activity.

Here we show that a major muscle specific isoform of the murine LINC complex protein SUN1 is required for efficient muscle regeneration. The nucleoplasmic domain of the isoform specifically binds to and inhibits Drosha, a key component of the microprocessor complex required for miRNA synthesis. Comparison of the miRNA profiles between wildtype and SUN1 null myotubes identified a cluster of miRNAs encoded by a non-translated retrotransposon-like one antisense (Rtl1as) transcript that are decreased in the WT myoblasts due to SUN1 inhibition of Drosha. One of these miRNAs miR-127 inhibits the translation of the Rtl1 sense transcript, that encodes the retrotransposon-like one protein (RTL1), which is also required for muscle regeneration and is expressed in regenerating/dystrophic muscle. The LINC complex may therefore regulate gene expression during muscle regeneration by controlling miRNA processing. This provides new insights into the molecular pathology underlying muscular dystrophies and how the LINC complex may regulate mechanosignaling.

TET3 prevents terminal differentiation of adult NSCs by a non-catalytic action at Snrpn.

Ten-eleven-translocation (TET) proteins catalyze DNA hydroxylation, playing an important role in demethylation of DNA in mammals. Remarkably, although hydroxymethylation levels are high in the mouse brain, the potential role of TET proteins in adult neurogenesis is unknown. We show here that a non-catalytic action of TET3 is essentially required for the maintenance of the neural stem cell (NSC) pool in the adult subventricular zone (SVZ) niche by preventing premature differentiation of NSCs into non-neurogenic astrocytes. This occurs through direct binding of TET3 to the paternal transcribed allele of the imprinted gene Small nuclear ribonucleoprotein-associated polypeptide N (Snrpn), contributing to transcriptional repression of the gene. The study also identifies BMP2 as an effector of the astrocytic terminal differentiation mediated by SNRPN. Our work describes a novel mechanism of control of an imprinted gene in the regulation of adult neurogenesis through an unconventional role of TET3.

Correction: Imprinting methylation in SNRPN and MEST1 in adult blood predicts cognitive ability.

[This corrects the article DOI: 10.1371/journal.pone.0211799.].

Imprinting methylation in SNRPN and MEST1 in adult blood predicts cognitive ability.

Genomic imprinting is important for normal brain development and aberrant imprinting has been associated with impaired cognition. We studied the imprinting status in selected imprints (H19, IGF2, SNRPN, PEG3, MEST1, NESPAS, KvDMR, IG-DMR and ZAC1) by pyrosequencing in blood samples from longitudinal cohorts born in 1936 (n = 485) and 1921 (n = 223), and anterior hippocampus, posterior hippocampus, periventricular white matter, and thalamus from brains donated to the Aberdeen Brain Bank (n = 4). MEST1 imprint methylation was related to childhood cognitive ability score (-0.416 95% CI -0.792,-0.041; p = 0.030), with the strongest effect evident in males (-0.929 95% CI -1.531,-0.326; p = 0.003). SNRPN imprint methylation was also related to childhood cognitive ability (+0.335 95%CI 0.008,0.663; p = 0.045). A significant association was also observed for SNRPN methylation and adult crystallised cognitive ability (+0.262 95%CI 0.007,0.517; p = 0.044). Further testing of significant findings in a second cohort from the same region, but born in 1921, resulted in similar effect sizes and greater significance when the cohorts were combined (MEST1; -0.371 95% CI -0.677,-0.065; p = 0.017; SNRPN; +0.361 95% CI 0.079,0.643; p = 0.012). For SNRPN and MEST1 and four other imprints the methylation levels in blood and in the five brain regions were similar. Methylation of the paternally expressed, maternally methylated genes SNRPN and MEST1 in adult blood was associated with cognitive ability in childhood. This is consistent with the known importance of the SNRPN containing 15q11-q13 and the MEST1 containing 7q31-34 regions in cognitive function. These findings, and their sex specific nature in MEST1, point to new mechanisms through which complex phenotypes such as cognitive ability may be inherited. These mechanisms are potentially relevant to both the heritable and non-heritable components of cognitive ability. The process of epigenetic imprinting-within SNRPN and MEST1 in particular-and the factors that influence it, are worthy of further study in relation to the determinants of cognitive ability.

Novel Primate Model of Serotonin Transporter Genetic Polymorphisms Associated with Gene Expression, Anxiety and Sensitivity to Antidepressants.

Genetic polymorphisms in the repeat upstream region of the serotonin transporter gene (SLC6A4) are associated with individual differences in stress reactivity, vulnerability to affective disorders, and response to pharmacotherapy. However, the molecular, neurodevelopmental and psychopharmacological mechanisms underlying the link between SLC6A4 polymorphisms and the emotionally vulnerable phenotype are not fully understood. Thus, using the marmoset monkey Callithrix jacchus we characterize here a new neurobiological model to help to address these questions. We first sequenced the marmoset SLC6A4 promoter and identified a double nucleotide polymorphism (-2053AC/CT) and two single-nucleotide polymorphisms (-2022C/T and -1592G/C) within the repeat upstream region. We showed their association with gene expression using in vivo quantitative PCR and with affective behavior using a primate test of anxiety (human intruder test). The low-expressing haplotype (AC/C/G) was linked with high anxiety while the high-expressing one (CT/T/C) was associated with an active coping strategy in response to threat. Pharmacological challenge with an acute dose of the selective serotonin reuptake inhibitor, citalopram, revealed a genotype-dependent behavioral response. While individuals homozygous for the high anxiety-related haplotype AC/C/G exhibited a dose-dependent, anxiogenic response, individuals homozygous for the low anxiety-related haplotype CT/T/C showed an opposing, dose-dependent anxiolytic effect. These findings provide a novel genetic and behavioral primate model to study the molecular, neurodevelopmental, and psychopharmacological mechanisms that underlie genetic variation-associated complex behaviors, with specific implications for the understanding of normal and abnormal serotonin actions and the development of personalized pharmacological treatments for psychiatric disorders.

Non-CG DNA methylation is a biomarker for assessing endodermal differentiation capacity in pluripotent stem cells.

Non-CG methylation is an unexplored epigenetic hallmark of pluripotent stem cells. Here we report that a reduction in non-CG methylation is associated with impaired differentiation capacity into endodermal lineages. Genome-wide analysis of 2,670 non-CG sites in a discovery cohort of 25 phenotyped human induced pluripotent stem cell (hiPSC) lines revealed unidirectional loss (Δß=13%, P<7.4 × 10(-4)) of non-CG methylation that correctly identifies endodermal differentiation capacity in 23 out of 25 (92%) hiPSC lines. Translation into a simplified assay of only nine non-CG sites maintains predictive power in the discovery cohort (Δß=23%, P<9.1 × 10(-6)) and correctly identifies endodermal differentiation capacity in nine out of ten pluripotent stem cell lines in an independent replication cohort consisting of hiPSCs reprogrammed from different cell types and different delivery systems, as well as human embryonic stem cell (hESC) lines. This finding infers non-CG methylation at these sites as a biomarker when assessing endodermal differentiation capacity as a readout.

A trans-homologue interaction between reciprocally imprinted miR-127 and Rtl1 regulates placenta development.

The paternally expressed imprinted retrotransposon-like 1 (Rtl1) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127- and Rtl1-deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1, mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.

In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism.

Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions.

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of "simple" cancer-associated chromosome deletions.

Epigenetic state and expression of imprinted genes in umbilical cord correlates with growth parameters in human pregnancy.

BACKGROUND: Genomic imprinting is a process causing genes to be expressed according to parental origin. Imprinting acts to coordinate fetal and prenatal growth, as well as control postnatal adaptations. Studies on human imprinting are confounded by tissue availability, sampling variability and limitations posed by tissue-specific expression and cellular heterogeneity within tissues. The human umbilical cord is an easily available, embryonic-derived fetal tissue with the potential to overcome many of these limitations. METHODS: In a sensitive, gene-specific quantitative expression analysis, we show for the first time robust imprinted gene expression combined with methylation analysis in cords isolated from Asian Chinese full-term births. RESULTS: Linear regression analyses revealed an inverse correlation between expression of pleckstrin homology-like domain, family A, member 2 (PHLDA2) with birth weight (BW). Furthermore, we observed significant down-regulation of the paternally expressed gene 10 (PEG10) in low BW babies compared to optimum BW babies. This change in PEG10 gene expression was accompanied by concomitant methylation alterations at the PEG10 promoter. CONCLUSIONS: These data are the first to demonstrate relative expression of an imprinted gene associated with epigenetic changes in non-syndromic fetal growth restriction in babies. They show that perturbed expression in compromised fetal growth may be associated with in utero modulation of the epigenetic state at the imprinting control regions and implicate specific imprinted genes as new biomarkers of fetal growth.

Trim28 is required for epigenetic stability during mouse oocyte to embryo transition.

Phenotypic variability in genetic disease is usually attributed to genetic background variation or environmental influence. Here, we show that deletion of a single gene, Trim28 (Kap1 or Tif1ß), from the maternal germ line alone, on an otherwise identical genetic background, results in severe phenotypic and epigenetic variability that leads to embryonic lethality. We identify early and minute epigenetic variations in blastomeres of the preimplantation embryo of these animals, suggesting that the embryonic lethality may result from the misregulation of genomic imprinting in mice lacking maternal Trim28. Our results reveal the long-range effects of a maternal gene deletion on epigenetic memory and illustrate the delicate equilibrium of maternal and zygotic factors during nuclear reprogramming.

Imprinted gene dosage is critical for the transition to independent life.

Neonatal survival in mammals is crucially dependent upon maintenance of body temperature. Neonatal body temperature is largely maintained by thermogenesis in brown adipose tissue (BAT). BAT develops perinatally in mice requiring integration of adipogenic and thermoregulatory gene pathways. We describe a regulatory mutation in the imprinted gene cluster on mouse chromosome 12 resulting in early postnatal lethality. Maternal inheritance of this mutation impairs the ability of young mice to maintain body temperature. While mechanisms of perinatal BAT development are well understood, our work highlights a second phase of BAT recruitment necessary to support small animals newly independent of the nest. We show that the imprinted delta-like homolog 1/preadipocyte factor (Dlk1/Pref1) and iodothyronine deiodinase type 3 (Dio3) functions converge on the development of brown fat at the transition to independent life. This shows that appropriate dosage control at imprinted loci can act as a critical determinant in postnatal survival during phases of physiological adaptation.

Status of genomic imprinting in epigenetically distinct pluripotent stem cells.

Mouse epiblast stem cells (EpiSCs) derived from postimplantation embryos are developmentally and functionally different from embryonic stem cells (ESCs) generated from blastocysts. EpiSCs require Activin A and FGF2 signaling for self-renewal, similar to human ESCs (hESCs), while mouse ESCs require LIF and BMP4. Unlike ESCs, EpiSCs have undergone X-inactivation, similar to the tendency of hESCs. The shared self-renewal and X-inactivation properties of EpiSCs and hESCs suggest that they have an epigenetic state distinct from ESCs. This hypothesis predicts that EpiSCs would have monoallelic expression of most imprinted genes, like that observed in hESCs. Here, we confirm this prediction. By contrast, we find that mouse induced pluripotent stem cells (iPSCs) tend to lose imprinting similar to mouse ESCs. These findings reveal that iPSCs have an epigenetic status associated with their pluripotent state rather than their developmental origin. Our results also reinforce the view that hESCs and EpiSCs are in vitro counterparts, sharing an epigenetic status distinct from ESCs and iPSCs.

A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints.

The mechanisms responsible for maintaining genomic methylation imprints in mouse embryos are not understood. We generated a knockout mouse in the Zfp57 locus encoding a KRAB zinc finger protein. Loss of just the zygotic function of Zfp57 causes partial neonatal lethality, whereas eliminating both the maternal and zygotic functions of Zfp57 results in a highly penetrant embryonic lethality. In oocytes, absence of Zfp57 results in failure to establish maternal methylation imprints at the Snrpn imprinted region. Intriguingly, methylation imprints are reacquired specifically at the maternally derived Snrpn imprinted region when the zygotic Zfp57 is present in embryos. This suggests that there may be DNA methylation-independent memory for genomic imprints. Zfp57 is also required for the postfertilization maintenance of maternal and paternal methylation imprints at multiple imprinted domains. The effects on genomic imprinting are consistent with the maternal-zygotic lethality of Zfp57 mutants.

Genomic imprinting at the mammalian Dlk1-Dio3 domain.

Genomic imprinting causes genes to be expressed or repressed depending on their parental origin. The majority of imprinted genes identified to date map in clusters and much of our knowledge of the mechanisms, function and evolution of imprinting have emerged from their analysis. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (Dlk1-Dio3) is located on distal mouse chromosome 12 and human chromosome 14. Its developmental importance is exemplified by severe phenotypes associated with altered dosage of these genes in mice and humans. The domain contains three imprinted protein-coding genes, Dlk1, Rtl1 and Dio3, expressed from the paternally inherited chromosome and several imprinted large and small noncoding RNA genes expressed from the maternally inherited homolog. Here, we discuss the function and regulation of imprinting at this domain.

High-frequency generation of viable mice from engineered bi-maternal embryos.

Mammalian development to adulthood typically requires both maternal and paternal genomes, because genomic imprinting places stringent limitations on mammalian development, strictly precluding parthenogenesis. Here we report the generation of bi-maternal embryos that develop at a high success rate equivalent to the rate obtained with in vitro fertilization of normal embryos. These bi-maternal mice developed into viable and fertile female adults. The bi-maternal embryos, distinct from parthenogenetic or gynogenetic conceptuses, were produced by the construction of oocytes from fully grown oocytes and nongrowing oocytes that contain double deletions in the H19 differentially methylated region (DMR) and the Dlk1-Dio3 intergenic germline-derived DMR. The results provide conclusive evidence that imprinted genes regulated by these two paternally methylated imprinting-control regions are the only paternal barrier that prevents the normal development of bi-maternal mouse fetuses to term.

Transpupillary thermotherapy-induced modification of angiogenesis- and coagulation-related gene expression in the rat posterior fundus.

PURPOSE: To study gene expression changes in the rat retina and choroid following transpupillary thermotherapy (TTT) and to identify molecular mechanisms that may enhance treatment of choroidal neovascularization, complicating age-related macular degeneration. METHODS: One fundus of Brown Norway rats was treated with an 810 nm diode laser while the contralateral fundus received no treatment. The mRNA was extracted and processed for cDNA microarray analysis. Genes with increased expression were validated by semiquantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR). RESULTS: Of the 14,815 cDNA elements on the array, 12 genes were up-regulated in TTT treated eyes. Upregulation of eight of these 12 genes could be verified by semiquantitative RT-PCR. The eight verified genes were EPCR, IL-1beta, MCP-1, TSP-1, Fgl, Asns, MT-2, and NMDMC, which included 4 angiogenesis- and coagulation-related genes. CONCLUSIONS: This study demonstrates upregulation of angiogenesis- and coagulation-related genes following TTT. The response profile and its temporal relationships provide insight into the molecular mechanisms that lead to vascular occlusion and antiangiogenesis induced by TTT.

Genomic imprinting contributes to thyroid hormone metabolism in the mouse embryo.

Many genes subject to genomic imprinting function in a number of endocrine/paracrine pathways that are important for normal mammalian development. Here, we show that an endocrine/paracrine pathway involving thyroid hormone metabolism is also regulated by imprinting. Thyroid hormone action depends on thyroid hormone receptors and their predominant ligand, 3,5,3'-triiodothyronine (T3). In vivo, thyroid hormone levels are maintained within the physiological range through the interaction of three iodothyronine deiodinases, D1, D2, and D3. D3 inactivates thyroxine (T4) and T3 by 5-deiodination, and the gene for this enzyme, Dio3, lies in the imprinted domain on human chromosome 14q32/distal mouse chromosome 12. Here, we report the imprinting of Dio3, which is expressed preferentially from the paternal allele. No differentially methylated region was identified in the CpG-island promoter, which is completely unmethylated. Localization of transcripts suggests that Dio3 may be exerting its function in both endocrine and autocrine/paracrine manners. An assay was developed for T3, and we show that its levels in maternal and paternal uniparental disomy (UPD) 12 fetuses are reciprocally affected. These results demonstrate that disruption of the imprinting status of Dio3 results in abnormal thyroid hormone levels and may contribute to the phenotypic abnormalities in UPD12 mice and UPD14 humans.