COOLAIR and PRC2 function in parallel to silence FLC during vernalization.

Noncoding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription (termed COOLAIR) interfaces with Polycomb Repressive Complex 2 (PRC2) silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). We use genetic and chromatin analyses on lines ineffective or hyperactive for the antisense pathway in combination with computational modeling to define the mechanisms underlying FLC repression. Our results show that FLC is silenced through pathways that function with different dynamics: a COOLAIR transcription-mediated pathway capable of fast response and in parallel a slow PRC2 switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the COOLAIR and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates by reduced dilution due to slow growth at low temperature. The parallel activities of the regulatory steps, and their control by temperature-dependent growth dynamics, create a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC.

Differential methylation of OPRK1 in borderline personality disorder is associated with childhood trauma.

According to a growing body of neurobiological evidence, the core symptoms of borderline personality disorder (BPD) may be linked to an opioidergic imbalance between the hedonic and stimulatory activity of mu opioid receptors (MOR) and the reward system inhibiting effects of kappa opioid receptors (KOR). Childhood trauma (CT), which is etiologically relevant to BPD, is also likely to lead to epigenetic and neurobiological adaptations by extensive activation of the stress and endogenous opioid systems. In this study, we investigated the methylation differences in the promoter of the KOR gene (OPRK1) in subjects with BPD (N = 47) and healthy controls (N = 48). Comparing the average methylation rates of regulatorily relevant subregions (specified regions CGI-1, CGI-2, EH1), we found no differences between BPD and HC. Analyzing individual CG nucleotides (N = 175), we found eight differentially methylated CG sites, all of which were less methylated in BPD, with five showing highly interrelated methylation rates. This differentially methylated region (DMR) was found on the falling slope (5') of the promoter methylation gap, whose effect is enhanced by the DMR hypomethylation in BPD. A dimensional assessment of the correlation between disease severity and DMR methylation rate revealed DMR hypomethylation to be negatively associated with BPD symptom severity (measured by BSL-23). Finally, analyzing the influence of CT on DMR methylation, we found DMR hypomethylation to correlate with physical and emotional neglect in childhood (quantified by CTQ). Thus, the newly identified DMR may be a biomarker of the risks caused by CT, which likely epigenetically contribute to the development of BPD.

Hypomethylated pollen bypasses the interploidy hybridization barrier in Arabidopsis.

Plants of different ploidy levels are separated by a strong postzygotic hybridization barrier that is established in the endosperm. Deregulated parent-of-origin specific genes cause the response to interploidy hybridizations, revealing an epigenetic basis of this phenomenon. In this study, we present evidence that paternal hypomethylation can bypass the interploidy hybridization barrier by alleviating the requirement for the Polycomb Repressive Complex 2 (PRC2) in the endosperm. PRC2 epigenetically regulates gene expression by applying methylation marks on histone H3. Bypass of the barrier is mediated by suppressed expression of imprinted genes. We show that the hypomethylated pollen genome causes de novo CHG methylation directed to FIS-PRC2 target genes, suggesting that different epigenetic modifications can functionally substitute for each other. Our work presents a method for the generation of viable triploids, providing an impressive example of the potential of epigenome manipulations for plant breeding.

High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis Endosperm.

Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.

EphrinA5 regulates cell motility by modulating Snhg15/DNA triplex-dependent targeting of DNMT1 to the Ncam1 promoter.

Cell-cell communication is mediated by membrane receptors and their ligands, such as the Eph/ephrin system, orchestrating cell migration during development and in diverse cancer types. Epigenetic mechanisms are key for integrating external "signals", e.g., from neighboring cells, into the transcriptome in health and disease. Previously, we reported ephrinA5 to trigger transcriptional changes of lncRNAs and protein-coding genes in cerebellar granule cells, a cell model for medulloblastoma. LncRNAs represent important adaptors for epigenetic writers through which they regulate gene expression. Here, we investigate a lncRNA-mediated targeting of DNMT1 to specific gene loci by the combined power of in silico modeling of RNA/DNA interactions and wet lab approaches, in the context of the clinically relevant use case of ephrinA5-dependent regulation of cellular motility of cerebellar granule cells. We provide evidence that Snhg15, a cancer-related lncRNA, recruits DNMT1 to the Ncam1 promoter through RNA/DNA triplex structure formation and the interaction with DNMT1. This mediates DNA methylation-dependent silencing of Ncam1, being abolished by ephrinA5 stimulation-triggered reduction of Snhg15 expression. Hence, we here propose a triple helix recognition mechanism, underlying cell motility regulation via lncRNA-targeted DNA methylation in a clinically relevant context.

VAL1 acts as an assembly platform co-ordinating co-transcriptional repression and chromatin regulation at Arabidopsis FLC.

Polycomb (PcG) silencing is crucial for development, but how targets are specified remains incompletely understood. The cold-induced Polycomb Repressive Complex 2 (PRC2) silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate PcG regulation. Association of the DNA binding protein VAL1 to FLC PcG nucleation regionis an important step. VAL1 co-immunoprecipitates APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and PRC1. Here, we show that ASAP and PRC1 are necessary for co-transcriptional repression and chromatin regulation at FLC. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulation increases at the FLC nucleation region during cold, but unlike the PRC2-delivered H3K27me3, does not spread across the locus. H2Aub thus involved in the transition to epigenetic silencing at FLC, facilitating H3K27me3 accumulation and long-term epigenetic memory. Overall, our work highlights the importance of VAL1 as an assembly platform co-ordinating activities necessary for epigenetic silencing at FLC.

Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds.

BACKGROUND: Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. RESULTS: cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was confirmed via allele-specific transcript analysis across a range of different accessions. Differentially methylated regions were identified adjacent to ATCDC48 and PDE120, which may represent candidate imprinting control regions. Finally, we demonstrate that expression levels of these three genes in vegetative tissues are MET1-dependent, while their uniparental maternal expression in the seed is not dependent on MET1. CONCLUSIONS: Using a cDNA-AFLP transcriptome profiling approach, we have identified three genes, ATCDC48, PDE120 and MS5-like which represent novel maternally expressed imprinted genes in the Arabidopsis thaliana seed. The extent of overlap between our cDNA-AFLP screen for maternally expressed imprinted genes, and other screens for imprinted and endosperm-expressed genes is discussed.

Code of Ethics and Conduct of the Brazilian Association of Embryologists - PRONÚCLEO.

For more than three decades, Brazilian Clinical Embryologists have been working without specific regulations and following the standards adopted by other healthcare professionals. This document aims to guide behavior and decision-making, while providing directions to embryologist with the purpose of aiding professionals involved with assisted reproduction procedures and their patients. The Code of Ethics and Conduct is an important breakthrough and the first step toward regulating Clinical Embryology as a profession.

Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis.

Chromosome dosage has substantial effects on reproductive isolation and speciation in both plants and animals, but the underlying mechanisms are largely obscure 1 . Transposable elements in animals can regulate hybridity through maternal small RNA 2 , whereas small RNAs in plants have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of long terminal repeat (LTR) retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21-22-nucleotide (nt) small RNAs in a dose-dependent fashion via RNA polymerase IV. We show that these epigenetically activated small interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (the 'triploid block'), and that natural variation for miR845 may account for 'endosperm balance' allowing the formation of triploid seeds. Targeting of the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.

Paternal easiRNAs regulate parental genome dosage in Arabidopsis.

The regulation of parental genome dosage is of fundamental importance in animals and plants, as exemplified by X-chromosome inactivation and dosage compensation. The 'triploid block' is a classic example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number1,2. This barrier acts in the embryo-nourishing endosperm tissue and induces the abortion of hybrid seeds through a yet unknown mechanism 3 . Here we show that depletion of paternal epigenetically activated small interfering RNAs (easiRNAs) bypasses the triploid block in response to increased paternal ploidy in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small-RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). Our data suggest that easiRNAs form a quantitative signal for paternal chromosome number and that their balanced dosage is required for post-fertilization genome stability and seed viability.

Hyaluronan-binding system for sperm selection enhances pregnancy rates in ICSI cycles associated with male factor infertility.

OBJECTIVES: The aim of the present study was to compare two procedures for sperm selection in ICSI cycles - conventional morphology sperm selection (ICSI-PVP) and chemical selection through Hyaluronan-treated petri dishes (PICSI), when male factor was associated. METHODS: The evaluated parameters were semen quality, fertilization and cleavage rates, chemical and clinical pregnancy rates, as well as abortion rate. Fifty-six ICSI cycles were included in this report, 19 cycles using PICSI and 37 using conventional ICSI. RESULTS: PICSI and ICSI showed, respectively, the following outcome: fertilization rates 71.93% (123/171) and 64.14% (127/198); cleavage rates 95.12% (117/123) and 95.27% (121/127); chemical pregnancy rates 63.15% (12/19) and 27.03% (10/37); clinical pregnancy rates 42.10% (8/19) and 16.21% (6/37); and abortion rates 33.33% (4/12) and 40.00% (4/10). According to both Fisher's Exact Test and Chi-square Test, chemical pregnancy (p = 0.05) and clinical pregnancy (p = 0.09) rates were significantly higher in the PICSI group. p values ≤ 0.05 were consider statistically significant. CONCLUSIONS: The present data indicates that ICSI cycles that used the PICSI technique had a considerably higher chance (≈5 fold) to achieve pregnancy than those who had sperm selected only by morphology assessment. Teratozoospermic patients were those who benefited most with PICSI. Therefore, the technique should be included in laboratory routine with low cost, avoiding the selection of immature sperm with increased rates of peroxidation and DNA fragmentation. Prospective and randomized studies should be applied to strengthen this suggestion.

Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana.

Genomic imprinting is an epigenetic phenomenon causing parent-of-origin specific differential expression of maternally and paternally inherited alleles. While many imprinted genes have been identified in plants, the functional roles of most of them are unknown. In this study, we systematically examine the functional requirement of paternally expressed imprinted genes (PEGs) during seed development in Arabidopsis thaliana. While none of the 15 analyzed peg mutants has qualitative or quantitative abnormalities of seed development, we identify three PEGs that establish postzygotic hybridization barriers in the endosperm, revealing that PEGs have a major role as speciation genes in plants. Our work reveals that a subset of PEGs maintains functional roles in the inbreeding plant Arabidopsis that become evident upon deregulated expression.

An imprinted gene underlies postzygotic reproductive isolation in Arabidopsis thaliana.

Postzygotic reproductive isolation in response to interploidy hybridizations is a well-known phenomenon in plants that forms a major path for sympatric speciation. A main determinant for the failure of interploidy hybridizations is the endosperm, a nutritious tissue supporting embryo growth, similar to the functional role of the placenta in mammals. Although it has been suggested that deregulated imprinted genes underpin dosage sensitivity of the endosperm, the molecular basis for this phenomenon remained unknown. In a genetic screen for suppressors of triploid seed abortion, we have identified the paternally expressed imprinted gene ADMETOS (ADM). Here, we present evidence that increased dosage of ADM causes triploid seed arrest. A large body of theoretical work predicted that deregulated imprinted genes establish the barrier to interploidy hybridization. Our study thus provides evidence strongly supporting this hypothesis and generates the molecular basis for our understanding of postzygotic hybridization barriers in plants.

Epigenetic mechanisms underlying genomic imprinting in plants.

Genomic imprinting, the differential expression of an autosomal gene that is dependent on its parent of origin, has independently evolved in flowering plants and mammals. In both of these organism classes, imprinting occurs in embryo-nourishing tissues-the placenta and the endosperm, respectively. It has been proposed that some imprinted genes control nutrient flow from the mother to the offspring. Genome-wide analyses of imprinted genes in plants have revealed that many imprinted genes are located in the vicinity of transposon or repeat sequences, implying that transposon insertions are associated with the evolution of imprinted loci. Imprinted expression of a number of genes is conserved between monocots and dicots, suggesting that long-term selection can maintain imprinted expression at some loci. In terms of epigenetic mechanisms, imprinted expression is largely controlled by an antagonistic action of DNA methylation and Polycomb group-mediated histone methylation in the vicinity of imprinted genes, whereby the position of such epigenetic modifications can determine whether a gene will be expressed mainly from either the maternally or paternally inherited alleles.

Tumor necrosis factor-á signaling cascades in apoptosis, necrosis, necroptosis and cell proliferation

Tumor necrosis factor-á (TNF-á) is a multifunctional cytokine involved in host defense, inflammation, apoptosis, autoimmunity, organogenesis and lymphoid microarchitecture. Many of these activities may be explained by the ability of this cytokine to induce distinct signal transduction pathways that recruit regulatory proteins involved in differentiation, cell death or cell proliferation. In this review, we discuss the contribution of caspases -3, -6, -7 and -8, and of cyclin-dependent kinases (CDKs), cyclin B and cyclin-dependent kinase inhibitors (CKI p21 and p27), as well as retinoblastoma tumor suppressor in the signaling cascades triggered by TNF-á to induce apoptosis, necrosis and cellular proliferation in the murine cell lines NIH3T3 and WEHI-164 and the human cervical carcinoma cell line HeLa-S3. Based on the findings of many literature reports and our own data, we discussed a model in which caspases are continuously activated throughout the cell cycle and kept at a critical threshold level by IAP (inhibitor of apoptosis) antagonists. Following the release of Smac/Diablo and HtrA2/OMI from mitochondria in response to diverse stimuli, this threshold is overcome and results in amplified caspase activation and cell death. An alternative, caspase-independent mechanism of cell death is induced in NIH3T3 fi broblasts by a combination of TNF and the pan-caspase inhibitor z-VADfmk. This cell death phenotype, known as necroptosis, displays some morphological features of apoptosis and necrosis. Although caspases are critical regulators of the TNF signaling pathway during cellular life and death, the mechanisms involved in the fine regulation of their dual effects remain to be fully elucidated.

Diagnóstico genético pré-implantacional: uma ferramenta importante para a rotina de fertilização in vitro? / Preimplantation genetic diagnosis: an important tool for the in vitro fertilization routine?

Erros cromossômicos numéricos são comuns durante as primeiras etapas do desenvolvimento embrionário humano, contribuem significativamente com processos de falta de implantação e são causadores da perda gestacional recorrente em pelo menos 50 por cento dos abortos ocorridos no primeiro trimestre. Tradicionalmente, a prevenção das anomalias genéticas cromossômicas em pacientes de alto risco é realizada por exames pré-natais, como a biópsia do vilo coriônico, aminiocentese e a cordocentese. Uma vez diagnosticada a anomalia, não existe tratamento eficaz para portadores de aberrações genéticas e a interrupção da gestação nestes casos ainda é ética e legalmente questionável. O diagnóstico genético pré-implantacional (DGPI) representa uma ferramenta valiosa aos casais de alto risco, por permitir a seleção de embriões saudáveis obtidos através de programas de fertilização in vitro antes de estes serem transferidos para um útero materno. Os primeiros relatos da utilização do DGPI datam da década de 90, quando eram utilizadas metodologias da reação em cadeia da polimerase para determinação do sexo do embrião, permitindo desta maneira transferir embriões que não fossem portadores de doenças ligadas ao cromossomo X. O DGPI é uma técnica extremamente eficaz, que analisa uma única célula do embrião que é biopsiado a partir do terceiro dia de desenvolvimento. Esta metodologia tem como finalidade identificar embriões gerados por processos de reprodução assistida, os quais sejam portadores de aberrações cromossômicas numéricas que envolvam os cromossomos X, Y, 13, 16, 18, 21 e 22. A metodologia mais utilizada para a realização do DGPI é a técnica de hibridização in situ, utilizando-se sondas fluorescentes para os cromossomos citados. Este é um método eficiente e que deve ser discutido com casais cuja idade da mulher seja acima dos 39 anos, casais com cariótipo alterado ou ainda casais com histórico familiar de presença de portadores de cromossomopatias. A eficiência...

The potential transmission of genetic disorders to the offspring has been a major problem for many couples when contemplating pregnancy. Numerical chromosomes errors are common during the first stages of the human embryonic development and contribute significantly with processes of implantation failure and it is also related to recurrent miscarriage, in at least 50 percent of the abortions occurred in the first trimester. Traditionally, the prevention of genetic chromosomal abnormalities in high risk patients is achieved by pre-natal examinations such as biopsy of the chorionic villi, aminiocentese of cordocentesis. Once diagnosed the genetic error, there is no effective treatment for patients with genetic aberrations and the interruption of pregnancy in these cases are ethically and legally questionable. The risk has been greatly decreases by the evaluation of the family history of the age of the mother, and the implementation of prenatal diagnosis in those couples in which the risk was increased compared to the general population. An alternative approach that is available with assisted reproductive technologies is the preimplantation genetic diagnosis (PGD), which it is possible to observe X, Y, 13, 16, 18, 21 and 22 chromosomes or to perform gene screen by PCR for knowing genetic diseases before the corresponding embryo is transferred to the uterus of the mother. PGD was first clinically applied in the early nineties, and was initially used in sexing cases for couples who were at risk of transmitting an X-linked recessive disorder. PGD involves the analysis of either polar bodies, extruded from oocytes during meiosis, or single cells (blastomeres) biopsied from embryos after fertilization. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization and polymerase chain reaction. The efficiency of the method is about 98 percent and its extremely invasive technique demands high level professional.