Variant PNLDC1, Defective piRNA Processing, and Azoospermia.

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

Tet3 ablation in adult brain neurons increases anxiety-like behavior and regulates cognitive function in mice.

TET3 is a member of the ten-eleven translocation (TET) family of enzymes which oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Tet3 is highly expressed in the brain, where 5hmC levels are most abundant. In adult mice, we observed that TET3 is present in mature neurons and oligodendrocytes but is absent in astrocytes. To investigate the function of TET3 in adult postmitotic neurons, we crossed Tet3 floxed mice with a neuronal Cre-expressing mouse line, Camk2a-CreERT2, obtaining a Tet3 conditional KO (cKO) mouse line. Ablation of Tet3 in adult mature neurons resulted in increased anxiety-like behavior with concomitant hypercorticalism, and impaired hippocampal-dependent spatial orientation. Transcriptome and gene-specific expression analysis of the hippocampus showed dysregulation of genes involved in glucocorticoid signaling pathway (HPA axis) in the ventral hippocampus, whereas upregulation of immediate early genes was observed in both dorsal and ventral hippocampal areas. In addition, Tet3 cKO mice exhibit increased dendritic spine maturation in the ventral CA1 hippocampal subregion. Based on these observations, we suggest that TET3 is involved in molecular alterations that govern hippocampal-dependent functions. These results reveal a critical role for epigenetic modifications in modulating brain functions, opening new insights into the molecular basis of neurological disorders.

TET3 controls the expression of the H3K27me3 demethylase Kdm6b during neural commitment.

The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.

Correction to: Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

The article Tet3 regulates cellular identity and DNA methylation in neural progenitor cells, written by Miguel R. Branco and C. Joana Marques, was originally published electronically on the publisher's internet portal.

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

Deregulation of imprinted genes expression and epigenetic regulators in placental tissue from intrauterine growth restriction.

PURPOSE: Intrauterine growth restriction (IUGR) is a fetal growth complication that can be caused by ineffective nutrient transfer from the mother to the fetus via the placenta. Abnormal placental development and function have been correlated with abnormal expression of imprinted genes, which are regulated by epigenetic modifications at imprinting control regions (ICRs). In this study, we analyzed the expression of imprinted genes known to be involved in fetal growth and epigenetic regulators involved in DNA methylation, as well as DNA methylation at the KvDMR1 imprinting control region and global levels of DNA hydroxymethylation, in IUGR cases. METHODS: Expression levels of imprinted genes and epigenetic regulators were analyzed in term placental samples from 21 IUGR cases and 9 non-IUGR (control) samples, by RT-qPCR. Additionally, KvDMR1 methylation was analyzed by bisulfite sequencing and combined bisulfite restriction analysis (COBRA) techniques. Moreover, global DNA methylation and hydroxymethylation levels were also measured. RESULTS: We observed increased expression of PHLDA2, CDKN1C, and PEG10 imprinted genes and of DNMT1, DNMT3A, DNMT3B, and TET3 epigenetic regulators in IUGR placentas. No differences in methylation levels at the KvDMR1 were observed between the IUGR and control groups; similarly, no differences in global DNA methylation and hydromethylation were detected. CONCLUSION: Our study shows that deregulation of epigenetic mechanisms, namely increased expression of imprinted genes and epigenetic regulators, might be associated with IUGR etiology. Therefore, this study adds knowledge to the molecular mechanisms underlying IUGR, which may contribute to novel prediction tools and future therapeutic options for the management of IUGR pregnancies.

Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation.

Methylation at the 5' position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

TET enzymes and DNA hydroxymethylation in neural development and function - how critical are they?

Epigenetic modifications of the genome play important roles in controlling gene transcription thus regulating several molecular and cellular processes. A novel epigenetic modification - 5-hydroxymethylcytosine (5hmC) - has been recently described and attracted a lot of attention due to its possible involvement in the active DNA demethylation mechanism. TET enzymes are dioxygenases capable of oxidizing the methyl group of 5-methylcytosines (5mC) and thus converting 5mC into 5hmC. Although most of the work on TET enzymes and 5hmC has been carried out in embryonic stem (ES) cells, the highest levels of 5hmC occur in the brain and in neurons, pointing to a role for this epigenetic modification in the control of neuronal differentiation, neural plasticity and brain functions. Here we review the most recent advances on the role of TET enzymes and DNA hydroxymethylation in neuronal differentiation and function.

The role of DNA hydroxymethylation and TET enzymes in placental development and pregnancy outcome.

The placenta is a temporary organ that is essential for supporting mammalian embryo and fetal development. Understanding the molecular mechanisms underlying trophoblast differentiation and placental function may contribute to improving the diagnosis and treatment of obstetric complications. Epigenetics plays a significant role in the regulation of gene expression, particularly at imprinted genes, which are fundamental in the control of placental development. The Ten-Eleven-Translocation enzymes are part of the epigenetic machinery, converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). DNA hydroxymethylation is thought to act as an intermediate in the DNA demethylation mechanism and potentially be a stable and functionally relevant epigenetic mark on its own. The role of DNA hydroxymethylation during differentiation and development of the placenta is not fully understood but increasing knowledge in this field will help to evaluate its potential role in pregnancy complications. This review focuses on DNA hydroxymethylation and its epigenetic regulators in human and mouse placental development and function. Additionally, we address 5hmC in the context of genomic imprinting mechanism and in pregnancy complications, such as intrauterine growth restriction, preeclampsia and pregnancy loss. The cumulative findings show that DNA hydroxymethylation might be important for the control of gene expression in the placenta and suggest a dynamic role in the differentiation of trophoblast cell types during gestation.

Novel Insights on the Role of Epigenetics in Androgen Receptor's Expression in Prostate Cancer.

The androgens/androgen receptor (AR) axis is the main therapeutic target in prostate cancer (PCa). However, while initially responsive, a subset of tumors loses AR expression through mechanisms putatively associated with epigenetic modifications. In this study, we assessed the link between the presence of CpG methylation in the 5'UTR and promoter regions of AR and loss of AR expression. Hence, we characterized and compared the methylation signature at CpG resolution of these regulatory regions in vitro, both at basal levels and following treatment with 5-aza-2-deoxycytidine (DAC) alone, or in combination with Trichostatin A (TSA). Our results showed heterogeneity in the methylation signature of AR negative cell lines and pinpointed the proximal promoter region as the most consistently methylated site in DU-145. Furthermore, this region was extremely resistant to the demethylating effects of DAC and was only significantly demethylated upon concomitant treatment with TSA. Nevertheless, no AR re-expression was detected at the mRNA or protein level. Importantly, after treatment, there was a significant increase in repressive histone marks at AR region 1 in DU-145 cells. Altogether, our data indicate that AR region 1 genomic availability is crucial for AR expression and that the inhibition of histone methyltransferases might hold promise for AR re-expression.

Tet3 Deletion in Adult Brain Neurons of Female Mice Results in Anxiety-like Behavior and Cognitive Impairments.

TET enzymes (TET1-3) are dioxygenases that oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and are involved in the DNA demethylation process. In line with the observed 5hmC abundance in the brain, Tet genes are highly transcribed, with Tet3 being the predominant member. We have previously shown that Tet3 conditional deletion in the brain of male mice was associated with anxiety-like behavior and impairment in hippocampal-dependent spatial orientation. In the current study, we addressed the role of Tet3 in female mice and its impact on behavior, using in vivo conditional and inducible deletion from post-mitotic neurons. Our results indicate that conditional and inducible deletion of Tet3 in female mice increases anxiety-like behavior and impairs both spatial orientation and short-term memory. At the molecular level, we identified upregulation of immediate-early genes, particularly Npas4, in both the dorsal and ventral hippocampus and in the prefrontal cortex. This study shows that deletion of Tet3 in female mice differentially affects behavioral dimensions as opposed to Tet3 deletion in males, highlighting the importance of studying both sexes in behavioral studies. Moreover, it contributes to expand the knowledge on the role of epigenetic regulators in brain function and behavioral outcome.

Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency: An Update on Genetic Analysis of CYP21A2 Gene.

Congenital Adrenal Hyperplasia is a group of genetic autosomal recessive disorders that affects adrenal steroidogenesis in the adrenal cortex. One of the most common defects associated with Congenital Adrenal Hyperplasia is the deficiency of 21-hydroxylase enzyme, responsible for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. The impairment of cortisol and aldosterone production is directly related to the clinical form of the disease that ranges from classic or severe to non-classic or mild late onset. The deficiency of 21-hydroxylase enzyme results from pathogenic variants on CYP21A2 gene that, in the majority of the cases, compromise enzymatic activity and are strongly correlated with the clinical severity of the disease. Due to the exceptionally high homology and proximity between the gene and the pseudogene, more than 90% of pathogenic variants result from intergenic recombination. Around 75% are deleterious variants transferred from the pseudogene by gene conversion, during mitosis. About 20% are due to unequal crossing over during meiosis and lead to duplications or deletions on CYP21A2 gene. Molecular genetic analysis of CYP21A2 variants is of major importance for confirmation of clinical diagnosis, predicting prognosis and for an appropriate genetic counselling. In this review we will present an update on the genetic analysis of CYP21A2 gene variants in CAH patients performed in our department.

Altered expression of epigenetic regulators and imprinted genes in human placenta and fetal tissues from second trimester spontaneous pregnancy losses.

Epigenetic mechanisms such as genomic imprinting have a fundamental role in embryo and fetal development. Hence, we here studied expression levels of epigenetic modifiers and imprinted genes in cases of ididopathic spontaneous abortion (SA). Thirty-five placental samples and 35 matched fetal tissues from second trimester SA were analysed; including 16 controls (placental and fetal infections as the known cause of spontaneous abortion) and 19 idiopathic SA cases. Transcript levels of epigenetic regulators and imprinted genes were measured by qRT-PCR and methylation at imprinted genes was studied by bisulfite genomic sequencing and MS-MLPA. Global DNA hydroxymethylation (5-hmC) levels were measured by an ELISA-based assay. We observed an upregulation of TET2 and TET3 in placental samples from idiopathic SA cases; however, no significant difference in global 5-hmC levels was observed. On the contrary, in fetal tissues, TET3 was markedly downregulated in idiopathic SA, showing an opposite trend to that observed in placental tissue. IGF2 and CDKN1C were upregulated and MEST downregulated in placentas from idiopathic SA cases; concordantly, IGF2 was also upregulated in fetal tissues from idiopathic SA cases. Although not reaching statistical significance, an increase in methylation levels of MEST, KvDMR1 and H19 DMRs was observed in idiopathic SA cases, concordantly with the observed changes in expression. Our study reveals, for the first time, deregulation of epigenetic modifiers and imprinted genes in both placental and fetal tissues from idiopathic SA cases in the second trimester of pregnancy, indicating a critical role during pregnancy.

TET enzymes in neurophysiology and brain function.

The dynamic nature of epigenetic DNA modifications is crucial for regulating gene expression in an experience-dependent manner and, thus, a potential mediator of neuronal plasticity and behavior. The discovery of the involvement of 5-hydroxymethylcytosine (5hmC) and Ten Eleven Translocation (TET) family of enzymes in the demethylation pathway uncovered a potential link between neuronal TET protein function and cognitive processes. In this review, we provide an overview on how profile of 5hmC and TET enzymes are powerful mechanisms to explain neuronal plasticity and long-term behaviors, such as cognition. More specifically, we discuss how the current knowledge integrates the function of each TET enzyme in neurophysiology and brain function.

Profiling DNA Methylation Based on Next-Generation Sequencing Approaches: New Insights and Clinical Applications.

DNA methylation is an epigenetic modification that plays a pivotal role in regulating gene expression and, consequently, influences a wide variety of biological processes and diseases. The advances in next-generation sequencing technologies allow for genome-wide profiling of methyl marks both at a single-nucleotide and at a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, coverage, and bioinformatics analysis. Thus, the selection of the most feasible method according with the project's purpose requires in-depth knowledge of those techniques. Currently, high-throughput sequencing techniques are intensively used in epigenomics profiling, which ultimately aims to find novel biomarkers for detection, diagnosis prognosis, and prediction of response to therapy, as well as to discover new targets for personalized treatments. Here, we present, in brief, a portrayal of next-generation sequencing methodologies' evolution for profiling DNA methylation, highlighting its potential for translational medicine and presenting significant findings in several diseases.

DNA demethylases: a new epigenetic frontier in drug discovery.

DNA methylation is one of the most extensively studied, and one of the most stable, of all epigenetic modifications. Two drugs that target DNA methyltransferase enzymes are licensed for clinical use in oncology but relatively little attention has focused on the enzymatic pathways by which DNA methylation can be reversed. Recent breakthroughs have identified at least two classes of enzymes that can achieve functional reversal. This review discusses the significance of DNA demethylation in a range of human diseases, the candidate proteins that mediate the demethylation and the opportunities and challenges in targeting these candidates to develop new therapeutics.

5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.

The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular mechanisms. We detect 5-hydroxymethylcytosine (5hmC) as a novel modification in mouse, bovine and rabbit zygotes. On zygotic development 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. A knockdown of the 5hmC generating dioxygenase Tet3 simultaneously affects the patterns of 5hmC and 5mC in the paternal pronucleus. This finding links the loss of 5mC to its conversion into 5hmC. The maternal pronucleus seems to be largely protected against this mechanism by PGC7/Dppa3/Stella, as in PGC7 knockout zygotes 5mC also becomes accessible to oxidation into 5hmC. In summary, our data suggest an important role of 5hmC and Tet3 for DNA methylation reprogramming processes in the mammalian zygote.

DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages.

Paternal imprinting marks were shown to be erased in the mouse primordial germ cells and progressively re-established throughout the male germ line development, starting in fetal prospermatogonia and continuing post-natally through the onset of meiosis. We here evaluated imprinting marks in human adult spermatogenic cells and analyzed mRNA and protein expression of DNA Methyltransferases (DNMTs). Spermatogonia A, primary and secondary spermatocytes, round spermatids and elongated spermatids/spermatozoa were isolated by micromanipulation from testicular biopsies of men with normal spermatogenesis. DNA methylation at two imprinted genes, H19 and MEST/PEG1, was analyzed using bisulphite genomic sequencing and DNMTs expression was determined by qRT-PCR and immunofluorescence. H19 was completely methylated at the spermatogonia stage in the analyzed individuals and MEST/PEG1 was completely demethylated, with the exception of few CpGs. The analysis of DNMT1, DNMT3A and 3B expression showed peaks of mRNA transcripts in primary spermatocytes and in mature ejaculated spermatozoa, with DNMT1 transcript level being the most abundant in all cell stages. Immunolocalization showed that DNMT proteins are present throughout the spermatogenic cycle, with stage-specific shuttling between the nucleus and cytoplasm. We conclude that, in humans, methylation imprints are established in spermatogonia A and are maintained in subsequent stages up to elongated spermatid/spermatozoa. Additionally, DNA methyltransferases are expressed throughout human spermatogenesis, possibly maintaining the methylation patterns in order to avoid the transmission of imprinting errors by the male gamete.

Methylation defects of imprinted genes in human testicular spermatozoa.

OBJECTIVE: To study the methylation imprinting marks of two oppositely imprinted genes, H19 and MEST/PEG1, in human testicular spermatozoa from azoospermic patients with different etiologies. Testicular spermatozoa are often used in intracytoplasmic sperm injection for treatment of male factor infertility, but the imprinting status of these cells is currently unknown. DESIGN: Experimental prospective study. SETTING: University research laboratory and private in vitro fertilization (IVF) clinic. PATIENT(S): A total of 24 men, five with anejaculation, five with secondary obstructive azoospermia, five with primary obstructive azoospermia, and nine with secretory azoospermia due to hypospermatogenesis. INTERVENTION(S): Spermatozoa were isolated by micromanipulation from testicular biopsies. MAIN OUTCOME MEASURE(S): DNA methylation patterns were analyzed using bisulfite genomic sequencing with cloning analysis. RESULT(S): We found H19 complete methylation was statistically significantly reduced in secretory azoospermic patients with hypospermatogenesis, with one patient presenting complete unmethylation. Hypomethylation also affected the CTCF-binding site 6, involved in regulation of IGF2 expression. Regarding the MEST gene, all patients presented complete unmethylation although this was statistically significantly reduced in the anejaculation group. CONCLUSION(S): Testicular spermatozoa from men with abnormal spermatogenesis carry methylation defects in the H19 imprinted gene which also affect the CTCF-binding site, further supporting an association between the occurrence of imprinting errors and disruptive spermatogenesis.