Dlk1-Dio3 Locus Research Articles

MiRNAs from the Dlk1-Dio3 locus and miR-224/452 cluster contribute to glioblastoma tumor heterogeneity

Glioblastoma is one of the most common and aggressive brain tumors and has seen few improvements in patient outcomes. Inter-tumor heterogeneity between tumors of different patients as well as intra-tumor heterogeneity of cells within the same tumor challenge the development of effective drugs. MiRNAs play an essential role throughout the developing brain and regulate many key genes involved in oncogenesis, yet their role in driving many of the processes underlying tumor heterogeneity remains unclear. In this study, we highlight miRNAs from the Dlk1-Dio3 and miR-224/452 clusters which may be expressed cell autonomously and have expression that is associated with cell state genes in glioblastoma, most prominently in neural progenitor-like and mesenchymal-like states respectively. These findings implicate these miRNA clusters as potential regulators of glioblastoma intra-tumoral heterogeneity and may serve as valuable biomarkers for cell state identification.

The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation.

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

Egr2 Deletion in Autoimmune-Prone C57BL6/lpr Mice Suppresses the Expression of Methylation-Sensitive Dlk1-Dio3 Cluster MicroRNAs.

We previously demonstrated that the upregulation of microRNAs (miRNAs) at the genomic imprinted Dlk1-Dio3 locus in murine lupus is correlated with global DNA hypomethylation. We now report that the Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice is hypomethylated, linking it to increased Dlk1-Dio3 miRNA expression. We evaluated the gene expression of methylating enzymes, DNA methyltransferases (DNMTs), and demethylating ten-eleven translocation proteins (TETs) to elucidate the molecular basis of DNA hypomethylation in lupus CD4+ T cells. There was a significantly elevated expression of Dnmt1 and Dnmt3b, as well as Tet1 and Tet2, in CD4+ T cells of three different lupus-prone mouse strains compared to controls. These findings suggest that the hypomethylation of murine lupus CD4+ T cells is likely attributed to a TET-mediated active demethylation pathway. Moreover, we found that deletion of early growth response 2 (Egr2), a transcription factor gene in B6/lpr mice markedly reduced maternally expressed miRNA genes but not paternally expressed protein-coding genes at the Dlk1-Dio3 locus in CD4+ T cells. EGR2 has been shown to induce DNA demethylation by recruiting TETs. Surprisingly, we found that deleting Egr2 in B6/lpr mice induced more hypomethylated differentially methylated regions at either the whole-genome level or the Dlk1-Dio3 locus in CD4+ T cells. Although the role of methylation in EGR2-mediated regulation of Dlk1-Dio3 miRNAs is not readily apparent, these are the first data to show that in lupus, Egr2 regulates Dlk1-Dio3 miRNAs, which target major signaling pathways in autoimmunity. These data provide a new perspective on the role of upregulated EGR2 in lupus pathogenesis.

Cross-Talk between miRNAs from the Dlk1-Dio3 Locus and Histone Methylation to Protect Male Cerebellum from Methyl Donor Deficiency.

Disruption of the one carbon metabolism during development, i.e., following a gestational vitamin B9 and B12 deficiencies, is involved in birth defects and brain development delay. Using a rat nutritional model, consisting of pups born to dams fed a vitamin B9 and B12 deficient diet (MDD), the study previously reports molecular and cellular alterations in the brain, in a sex dependent manner, with females being more affected than males. The study hypothesizes that epigenetic modifications could participate in the sex differences is observed. The study investigates lysine methylation of histones and expression of microRNAs in the cerebellum of MDD male and female pups. The study reports a differential regulation of H3K36Me2 and H4K20Me3 between males and females, in response to MDD. Moreover, distinct regulation of Kmt5b and Kdm2a expression by miR-134-5p and miR-369-5p from the Dlk1-Dio3 locus, contributes to the maintenance of expression of genes involved in synaptic plasticity. These results could explain the neuroprotection to MDD that male pups display. The work will contribute to the understanding of the consequences of vitamin starvation on brain development, as well as how the epigenome is affected by one carbon metabolism disruption.

Time-resolved Small-RNA Sequencing Identifies MicroRNAs Critical for Formation of Embryonic Stem Cells from the Inner Cell Mass of Mouse Embryos.

Cells of the inner cell mass (ICM) acquire a unique ability for unlimited self-renewal during transition into embryonic stem cells (ESCs) in vitro, while preserving their natural multi-lineage differentiation potential. Several different pathways have been identified to play roles in ESC formation but the function of non-coding RNAs in this process is poorly understood. Here, we describe several microRNAs (miRNAs) that are crucial for efficient generation of mouse ESCs from ICMs. Using small-RNA sequencing, we characterize dynamic changes in miRNA expression profiles during outgrowth of ICMs in a high-resolution, time-course dependent manner. We report several waves of miRNA transcription during ESC formation, to which miRNAs from the imprinted Dlk1-Dio3 locus contribute extensively. In silico analyses followed by functional investigations reveal that Dlk1-Dio3 locus-embedded miRNAs (miR-541-5p, miR-410-3p, and miR-381-3p), miR-183-5p, and miR-302b-3p promote, while miR-212-5p and let-7d-3p inhibit ESC formation. Collectively, these findings offer new mechanistic insights into the role of miRNAs during ESC derivation.

Analysis of miRNA Expression from the DLK1-DIO3 Locus in CD4+ and CD14+ Cells in Relapsing-Remitting Multiple Sclerosis

Analysis of miRNA Expression from the DLK1-DIO3 Locus in CD4+ and CD14+ Cells in Relapsing-Remitting Multiple Sclerosis

Effect of epigenetic activating of Dlk1-Dio3 imprinted cluster on miR-370 expression due to folate deficiency during nerve development

Proper Dlk1-Dio3 imprinting plays a critical role in embryogenesis, and folic acid deficiency may affect the imprinting of this locus through epigenetic regulation. However, whether and how folic acid directly impacts the imprinting status of Dlk1-Dio3 to affect neural development remain unclear. Here, we found decreased IG-DMR (intergenic -differentially methylated regions) methylation in the folate-deficient encephalocele in humans, suggesting that abnormal Dlk1-Dio3 imprinting status is related to neural tube defects (NTDs) caused by folate deficiency. Similar results were obtained with folate-deficient embryonic stem cells. By miRNA chip analysis, folic acid deficiency led to changes in multiple miRNAs, including the upregulation of 15 miRNAs located in the Dlk1-Dio3 locus. Real-time PCR confirmed that seven of these miRNAs were upregulated, especially miR-370. In contrast to normal embryonic development, in which expression of miR-370 is highest at E9.5, the abnormally high and sustained expression of miRNA-370 in folate-deficient E13.5 embryos may contribute to NTDs. In addition, we found that DNMT3A (de novo DNA methyltransferases 3A) is a direct target gene of miR-370 in neural cells, and DNMT3A participates in the role of miR-370 in inhibiting cell migration. Finally, in the folate-deficient mouse model, Dlk1-Dio3 epigenetic activation was found in fetal brain tissue, along with the upregulation of miR-370 and the downregulation of DNMT3A. Collectively, our findings demonstrate a pivotal role of folate in the epigenetic regulation of Dlk1-Dio3 imprinting during neurogenesis, revealing an elegant mechanism for the activation of Dlk1-Dio3 locus miRNAs in folic acid deficiency.

Unraveling the epitranscriptome of small non-coding RNAs in vascular cells

Unraveling the epitranscriptome of small non-coding RNAs in vascular cells

C/D box snoRNA SNORD113-6 guides 2′-O-methylation and protects against site-specific fragmentation of tRNALeu(TAA) in vascular remodeling

C/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascular disease. None of these snoRNAs has any known targets yet except for one, AF357425/SNORD113-6. We previously showed that this snoRNA targets mRNAs of the integrin signaling pathway and affects arterial fibroblast function. Here, we aimed to identify whether AF357425/SNORD113-6 can also target small RNAs. We overexpressed or inhibited AF357425 in murine fibroblasts and performed small RNA sequencing. Expression of transfer (t)RNA fragments (tRFs) was predominantly regulated. Compared with overexpression, AF357425 knockdown led to an overall decrease in tRFs but with an enrichment in smaller tRFs (<30 nucleotides). We focused on tRNA leucine anti-codon TAA (tRNALeu(TAA)), which has a conserved predicted binding site for AF357425/SNORD113-6. Adjacent to this site, the tRNA is cleaved to form tRFLeu 47–64 in both primary murine and human fibroblasts and in intact human arteries. We show that AF357425/SNORD113-6 methylates tRNALeu(TAA) and thereby prevents the formation of tRFLeu 47–64. Exposing fibroblasts to oxidative or hypoxic stress increased AF357425/SNORD113-6 and tRNALeu(TAA) expression, but AF357425/SNORD113-6 knockdown did not increase tRFLeu 47–64 formation under stress even further. Thus, independent of cellular stress, AF357425/SNORD113-6 protects against site-specific fragmentation of tRNALeu(TAA) via 2′O-ribose-methylation.

Involvement of PGC7 and UHRF1 in the regulation of DNA methylation of the IG-DMR in the imprinted Dlk1-Dio3 locus.

The gene dosage at the imprinted Dlk1-Dio3 locus is critical for cell growth and development. A relatively high gene expression within the Dlk1-Dio3 region, especially the active expression of Gtl2, has been identified as the only reliable marker for cell pluripotency. The DNA methylation state of the IG-DNA methylated regions (DMR), which is located upstream of the Gtl2 gene, dominantly contributes to the control of gene expression in the Dlk1-Dio3 locus. However, the precise mechanism underlying the regulation of DNA methylation in the IG-DMR remains largely unknown. Here, we use the F9 embryonal carcinoma cell line, a low pluripotent cell model, to identify the mechanism responsible for DNA methylation in the IG-DMR, and find that the interaction of PGC7 with UHRF1 is involved in maintaining DNA methylation and inducing DNA hypermethylation in the IG-DMR region. PGC7 and UHRF1 cooperatively bind in the IG-DMR to regulate the methylation of DNA and histones in this imprinted region. PGC7 promotes the recruitment of DNMT1 by UHRF1 to maintain DNA methylation in the IG-DMR locus. The interaction between PGC7 and UHRF1 strengthens their binding to H3K9me3 and leads to further enrichment of H3K9me3 in the IG-DMR by recruiting the specific histone methyltransferase SETDB1. Consequently, the abundance of H3K9me3 promotes DNMT3A to bind to the IG-DMR and increases DNA methylation level in this region. In summary, we propose a new mechanism of DNA methylation regulation in the IG-DMR locus and provide further insight into the understanding of the difference in Gtl2 expression levels between high and low pluripotent cells.

Male-specific coordinated changes in expression of miRNA genes, but not other genes within the DLK1-DIO3 locus in multiple sclerosis

The role of miRNAs, small non-coding regulatory RNAs, in the molecular mechanisms of multiple sclerosis (MS) development has been intensively studied. MiRNAs tend to be clustered within imprinted regions, and the largest number of miRNA genes is observed in the DLK1-DIO3 locus. Earlier using RNA-seq we identified sex-specific upregulation of the set of miRNA genes from this locus in peripheral blood mononuclear cells (PBMC) of treatment-naive relapsing-remitting MS (RRMS) patients. In the present study we set up to independently investigate the expression of a vast array of genes present in the DLK1-DIO3 imprinted locus. First, we analyzed the expression of miRNA genes, which levels in RRMS were mostly inconsistent based on RNA-seq data and not previously explored using qPCR. We identified that all selected miRNAs – miR-337-3p and -665 from 14q32.2 cluster and miR-370c, -380, -494, -654-3p, -300, -539, -668, and -323b-5p – were upregulated in MS men, but not women when compared to controls, regardless of conflicting RNA-seq data. The expression of miRNAs from the DLK1-DIO3 locus was highly correlated, indicating the existence of a common regulatory mechanism(s) that controls miRNA expression, regardless of the position of their genes within this region. Second, we performed the expression analysis of non-miRNA genes within the locus. The genes encoding proteins (DLK1, DIO3, RTL1), long non-coding RNAs (MEG3, MEG8, and MEG9) and small nucleolar RNAs (SNORD112, SNORD113-5, SNORD113-7, SNORD114-3, SNORD114-8, SNORD114-19) were not dysregulated in RRMS both in men and women. DNA methylation analysis of selected CpG sites within the differentially methylated regions IG-DMR, MEG3-DMR, and MEG8-DMR of the DLK1-DIO3 imprinted locus pointed out that they were not involved in the regulation of miRNA gene expression in RRMS, at least in PBMC population. The question of whether the observed changes in expression of miRNA genes (given that there is a constant expression of other non-miRNA genes of the DLK1-DIO3 locus) are involved in the development of RRMS or are they a consequence of the disease progress, remains open and needs further investigation.

Subnuclear localisation is associated with gene expression more than parental origin at the imprinted Dlk1-Dio3 locus.

At interphase, de-condensed chromosomes have a non-random three-dimensional architecture within the nucleus, however, little is known about the extent to which nuclear organisation might influence expression or vice versa. Here, using imprinting as a model, we use 3D RNA- and DNA-fluorescence-in-situ-hybridisation in normal and mutant mouse embryonic stem cell lines to assess the relationship between imprinting control, gene expression and allelic distance from the nuclear periphery. We compared the two parentally inherited imprinted domains at the Dlk1-Dio3 domain and find a small but reproducible trend for the maternally inherited domain to be further away from the periphery however we did not observe an enrichment of inactive alleles in the immediate vicinity of the nuclear envelope. Using Zfp57KO ES cells, which harbour a paternal to maternal epigenotype switch, we observe that expressed alleles are significantly further away from the nuclear periphery.However, within individual nuclei, alleles closer to the periphery are equally likely to be expressed as those further away. In other words, absolute position does not predict expression. Taken together, this suggests that whilst stochastic activation can cause subtle shifts in localisation for this locus, there is no dramatic relocation of alleles upon gene activation. Our results suggest that transcriptional activity, rather than the parent-of-origin, defines subnuclear localisation at an endogenous imprinted domain.

Pan-cancer analysis of microRNA expression profiles highlights microRNAs enriched in normal body cells as effective suppressors of multiple tumor types: A study based on TCGA database

BackgroundMicroRNAs (miRNAs) are frequently deregulated in various types of cancer. While antisense oligonucleotides are used to block oncomiRs, delivery of tumour-suppressive miRNAs holds great potential as a potent anti-cancer strategy. Here, we aim to determine, and functionally analyse, miRNAs that are lowly expressed in various types of tumour but abundantly expressed in multiple normal tissues.MethodsThe miRNA sequencing data of 14 cancer types were downloaded from the TCGA dataset. Significant differences in miRNA expression between tumor and normal samples were calculated using limma package (R programming). An adjusted p value < 0.05 was used to compare normal versus tumor miRNA expression profiles. The predicted gene targets were obtained using TargetScan, miRanda, and miRDB and then subjected to gene ontology analysis using Enrichr. Only GO terms with an adjusted p < 0.05 were considered statistically significant. All data from wet-lab experiments (cell viability assays and flow cytometry) were expressed as means ± SEM, and their differences were analyzed using GraphPad Prism software (Student’s t test, p < 0.05).ResultsBy compiling all publicly available miRNA profiling data from The Cancer Genome Atlas (TCGA) Pan-Cancer Project, we reveal a small set of tumour-suppressing miRNAs (which we designate as ’normomiRs’) that are highly expressed in 14 types of normal tissues but poorly expressed in corresponding tumour tissues. Interestingly, muscle-enriched miRNAs (e.g. miR-133a/b and miR-206) and miRNAs from DLK1-DIO3 locus (e.g. miR-381 and miR-411) constitute a large fraction of the normomiRs. Moreover, we define that the CCCGU motif is absent in the oncomiRs’ seed sequences but present in a fraction of tumour-suppressive miRNAs. Finally, the gain of function of candidate normomiRs across several cancer cell types indicates that miR-206 and miR-381 exert the most potent inhibition on multiple cancer types in vitro.ConclusionOur results reveal a pan-cancer set of tumour-suppressing miRNAs and highlight the potential of miRNA-replacement therapies for targeting multiple types of tumour.

Changes in expression of miRNAs from the DLK1-DIO3 locus are characteristic of relapsing-remitting multiple sclerosis regardless of the disease activity

The role of miRNAs – small, regulatory, noncoding RNAs – in the multiple sclerosis (MS) development is being intensively investigated. Previously, we the first who observed a significant increase in the expression of 26 microRNA genes localized in the DLK1-DIO3 locus in men with relapsing-remitting MS (RRMS) in peripheral blood mononuclear cells (PBMCs), using RNA sequencing.Objective: to evaluate the possible involvement of miRNAs in the regulation of the activity of the pathological process in RRMS by comparing the miRNA genes expression levels from this locus in patients in remission, relapse, and healthy individuals, separately for men and women.Patients and methods. Analysis of miR-431-5p, miR-127-3p, miR-379, miR-376c, miR-381, miR-410 and miR-656-3p microRNA expression was performed by reverse transcription and subsequent real-time polymerase chain reaction in PBMCs of 16 patients in the relapse stage, 20 patients in remission who did not receive immunomodulatory drugs, and 20 healthy individuals.Results and discussion. The expression levels of all studied miRNAs did not differ in patients in remission and relapse stages, in men and women. At the same time, men (in remission and relapse) showed a significant increase in the levels of all miRNAs compared with healthy men; in women, no changes in expression were observed. A high level of correlation of miRNA expression from the DLK1-DIO3 locus was shown in both patients and healthy individuals from the control group, regardless of gender. Coexpression was observed not only for miRNA genes from the same cluster (14q32.2 or 14q32.31), but also for genes from different clusters.Conclusion. The miRNAs genes from the DLK1-DIO3 locus are involved in the pathophysiology of RMS onset, but not in the processes associated with the transition from remission to relapses. The high consistency of miRNA expression, regardless of the localization of their genes within this region, suggests the presense of a common mechanism that regulates their transcription.

Expression analysis of miRNAs from the DLK1-DIO3 locus in CD4+ and CD14+ cells in patients with relapsing-remitting multiple sclerosis

To analyze the expression of several previously unstudied miRNAs from the DLK1-DIO3 locus in peripheral blood mononuclear cells (PBMC) and in CD14+ and CD4+ cell subpopulations in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals. MiRNA expression analysis was performed in PBMC, CD14+, and CD4+ cells of 14 patients who did not take immunomodulatory drugs, and 14 individuals without autoimmune and inflammatory diseases by reverse transcription followed by real-time PCR. Expression levels of 10 miRNAs selected based on different criteria were significantly higher in PBMC in men with RRMS compared to healthy men; in women, their expression did not change in a similar comparison. No mass unidirectional changes in the expression of these miRNAs in RRMS men were observed in CD14+ and CD4+ cells. High consistency in the expression of miRNA pairs was also not found. The analysis of PBMC showed the involvement of 10 more miRNAs encoded by genes from the DLK1-DIO3 locus in the development of RRMS. The main observed effect of the increase in the expression levels of these miRNAs in men with RRMS compared with healthy men is not achieved due to the contribution of CD14+ and CD4+ cells. This opens up possibilities for studying the involvement of miRNAs in other PBMC subpopulations in the pathological processes in RRMS.

Tyrosine kinases: a target of epigenetic influences and a new direction in the treatment of multiple sclerosis

Studies have shown that tyrosine kinases can be important in the pathogenesis of multiple sclerosis (MS) through the regulatory network of microRNAs. A microRNA network-based analysis revealed 17 receptor pathways activated by tyrosine kinase and regulated by microRNAs encoded at the DLK1-DIO3 locus on chromosome 14. Tyrosine kinases are actively involved in the epigenetic regulation of the pathological process in MS with the participation of a microRNA network and thus attract attention as a target for the development of new ways for the treatment of MS. The greatest attention was attracted by Bruton tyrosine kinase inhibitors (TKBi), which showed their ability to suppress the activity of autoimmune inflammatory lesions in the MS model - experimental autoimmune encephalomyelitis (EAE). This is due to the influence of TRB on the activity of B cells, which have a critical role in the development of the pathological process. The review discusses the types of TRBi, the features of their action in EAE and the results of the first studies in MS.

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus.

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.

Two sides of the Dlk1-Dio3 story in imprinting.

Loss of imprinting (LOI) at the Dlk1-Dio3 locus is linked to Kagami-Ogata and Temple syndromes, and to cancer, but molecular mechanisms that prevent LOI are under-studied. In this issue of Developmental Cell, Aronson etal. demarcate the bipartite regulation of the Dlk1-Dio3 imprinting control region (ICR) IG-DMR, which maintains locus imprinting.

C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2'O-ribose methylation.

We have previously shown that C/D box small nucleolar RNAs (snoRNAs) transcribed from the DLK1-DIO3 locus on human chromosome 14 (14q32) are associated with cardiovascular disease. DLK1-DIO3 snoRNAs are ‘orphan snoRNAs’ that have no known targets. We aimed to identify RNA targets and elucidate the mechanism-of-action of human SNORD113-6 (AF357425 in mice). As AF357425-knockout cells were non-viable, we induced overexpression or inhibition of AF357425 in primary murine fibroblasts and performed RNA-Seq. We identified several pre-mRNAs with conserved AF357425/SNORD113-6 D′-seed binding sites in the last exon/3′ untranslated region (3′UTR), which directed pre-mRNA processing and splice-variant-specific protein expression. We also pulled down the snoRNA-associated methyltransferase fibrillarin from AF357425-High versus AF357425-Low fibroblast lysates, followed by RNA isolation, ribosomal RNA depletion and RNA-Seq. Identifying mostly mRNAs, we subjected these to PANTHER pathway analysis and observed enrichment for genes in the integrin pathway. We confirmed 2′O-ribose methylation in six integrin pathway mRNAs (MAP2K1, ITGB3, ITGA7, PARVB, NTN4 and FLNB). Methylation and mRNA expressions were decreased while mRNA degradation was increased under AF357425/SNORD113-6 inhibition in both murine and human primary fibroblasts, but effects on protein expression were more ambiguous. Integrin signalling is crucial for cell–cell and cell–matrix interactions, and correspondingly, we observed altered human primary arterial fibroblast function upon SNORD113-6 inhibition.

Human placental piwi-interacting RNA transcriptome is characterized by expression from the DLK1-DIO3 imprinted region

The placenta is vital to embryonic development and requires a finely-tuned pattern of gene expression, achieved in part by its unique epigenetic landscape. Piwi-interacting RNAs (piRNAs) are a class of small-non-coding RNA with established roles as epigenetic regulators of gene expression, largely via methylation of targeted DNA sequences. The expression of piRNAs have mainly been described in germ cells, but a fraction have been shown to retain expression in adult somatic tissues. To aid in understanding the contribution of these regulators in the placenta, we provide the first description of the piRNA transcriptome in human placentas. We find 297 piRNAs to be preferentially expressed in the human placenta, a subset of which are expressed at higher levels relative to testes samples. We also observed a large proportion of placental piRNAs to be expressed from a single locus, as distinct from canonical cluster locations associated with transposable element silencing. Finally, we find that 15 of the highest-expressed placental piRNAs maps to the DLK1-DIO3 locus, suggesting a link to placental biology. Our findings suggest that piRNAs could contribute to the molecular networks defining placental function in humans, and a biological impact of piRNA expression beyond germ cells.