PRDM14 Expression Research Articles

Inhibition of protein kinase C increases Prdm14 level to promote self-renewal of embryonic stem cells through reducing Suv39h-induced H3K9 methylation

Inhibition of protein Kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA- sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, LC‒MS profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize di- and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, ChIP assays showed that Go6983 treatment led to decreased enrichment of di- and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.

Treatment of primary and metastatic breast and pancreatic tumors upon intravenous delivery of a PRDM14-specific chimeric siRNA/nanocarrier complex.

PRDM14 is highly expressed in several cancers but is not detected in normal tissues. It confers cancer stem cell-like properties, including chemoresistance and distant metastasis, to cancer cells. Herein, we aimed to develop a highly effective therapy against advanced stage cancer based on intravenously delivered PRDM14-targeted siRNA. First, we examined PRDM14 expression and gene amplification in breast and pancreatic tumors and cell lines. PRDM14 was expressed in breast cancer, including the triple-negative subtype, and pancreatic cancer. PRDM14 was amplified in 23.8% of patients with PRDM14+ breast cancer. Next, we investigated the inoculated tumor growth and distant metastasis following PRDM14 depletion by administering mice with PRDM14-specific chimeric siRNA combined with a novel branched PEGylated poly-L-ornithine (PLO)-based intravenous drug delivery system, designated PRDM14 unit polyion complex (uPIC) (n = 6/group). Inhibition of PRDM14 expression with PRDM14 uPIC by systemic intravenous injection effectively reduced tumor size and metastasis in vivo, thereby improving survival. Finally, pharmacokinetic/toxicokinetic analyses were performed on PRDM14 uPIC, which was intravenously administered to rats (n = 10-15/group) and cynomolgus monkeys (n = 3-5/group), twice weekly for 4 weeks. This revealed that PRDM14 uPIC was relatively nontoxic and the siRNA exposure in serum was greater than that predicted by the administered dose ratio when delivered as a uPIC. Taken together, our study indicated that PRDM14 uPIC is highly effective in suppressing malignant features of solid cancers and does not cause severe toxicity, making it a promising therapeutic agent for cancer treatment.

MLL1 combined with GSK3 and MAP2K inhibition improves the development of in vitro-fertilized embryos

The MM-102 compound prevents the interaction between mixed lineage leukemia 1 (MLL1) and WD Trp-Asp repeat domain 5 (WDR5) and results in the inhibition of MLL1 H3K4 histone methyltransferase (HMT) activity. The inhibition of the FGFR signaling pathway and activation of the WNT pathway by small molecule inhibitors (known as 2i) improves blastocyst development. However, studies on the effects of MLL1 combined with GSK3 and MAP2K inhibition (3i) on the development of embryos have not been reported. Our results show that 3i improves bovine and mouse IVF development only when added at the appropriate time point and affects ICM-related gene (OCT4, SOX2 and NANOG) expression in a concentration-dependent manner. 3i increases the expression of blastocyst-related genes such as PRDM14, KLF4 and KLF17 and decreases the expression of the de novo DNA methyltransferase genes DNMT3L and DNMT1 in bovines, but increases Prdm14, Stella, Klf2 and Klf4 expression and significantly decreases Dnmt3l, Dnmt3b, and Dnmt1 expression in mice. The analysis of transcription data showed that the expression of DNMTs increases slightly later than that of PRDM14 during embryo development, which indicates that PRDM14 is the upstream regulator. 3i upregulates PRDM14 and then downregulates DNMTs to affect IVF embryo development. When 3i-treated mouse embryos were transplanted, the morphology and body weight of the offspring were not significantly different from those of the control group. These offspring were as fertile as normal mice. 3i improves the development of bovine and mouse IVF embryos but does not affect the quality of the embryos. The application of 3i provides a new method for improving IVF embryo production in domestic animals.

PRDM14 promotes malignant phenotype and correlates with poor prognosis in colorectal cancer.

Emerging evidence suggests that stemness in cancer cells is a cause of drug resistance or metastasis and is an important therapeutic target. PR [positive regulatory domain I-binding factor 1 (PRDI-BF1) and retinoblastoma protein-interacting zinc finger gene (RIZ1)] domain containing 14 (PRDM14), that regulates pluripotency in primordial germ cell, has reported the overexpression and function of stemness in various malignancies, suggesting it as the possible therapeutic target. However, to our knowledge, there have been no reports on the expression and function of PRDM14 in colorectal cancer (CRC). Therefore, we investigated the expression and the role of PRDM14 in CRC. We performed immunohistochemistry evaluations and assessed PRDM14 expression on 414 primary CRC specimens. Colon cancer cell lines were subjected to functional and stemness assays in vitro and in vivo. We found that PRDM14 positive staining exhibited heterogeneity in the CRC primary tumor, especially at the tumor invasion front. The aberrant expression of PRDM14 at the invasion front was associated with lymph node metastasis and disease stage in patients with CRC. Furthermore, the multivariate analysis revealed high PRDM14 expression as an independent prognostic factor in the patients with Stage III CRC. Overexpression of PRDM14 enhanced the invasive, drug-resistant and stem-like properties in colon cancer cells in vitro and tumorigenicity in vivo. Our findings suggest that PRDM14 is involved in progression and chemoresistance of CRC, and is a potential prognostic biomarker and therapeutic target in the CRC patients.

The Pluripotency Regulator PRDM14 Requires Hematopoietic Regulator CBFA2T3 to Initiate Leukemia in Mice.

PR domain-containing 14 (Prdm14) is a pluripotency regulator central to embryonic stem cell identity and primordial germ cell specification. Genomic regions containing PRDM14 are often amplified leading to misexpression in human cancer. Prdm14 expression in mouse hematopoietic stem cells (HSC) leads to progenitor cell expansion prior to the development of T-cell acute lymphoblastic leukemia (T-ALL), consistent with PRDM14's role in cancer initiation. Here, we demonstrate mechanistic insight into PRDM14-driven leukemias in vivo. Mass spectrometry revealed novel PRDM14-protein interactions including histone H1, RNA-binding proteins, and the master hematopoietic regulator CBFA2T3. In mouse leukemic cells, CBFA2T3 and PRDM14 associate independently of the related ETO family member CBFA2T2, PRDM14's primary protein partner in pluripotent cells. CBFA2T3 plays crucial roles in HSC self-renewal and lineage commitment, and participates in oncogenic translocations in acute myeloid leukemia. These results suggest a model whereby PRDM14 recruits CBFA2T3 to DNA, leading to gene misregulation causing progenitor cell expansion and lineage perturbations preceding T-ALL development. Strikingly, Prdm14-induced T-ALL does not occur in mice deficient for Cbfa2t3, demonstrating that Cbfa2t3 is required for leukemogenesis. Moreover, T-ALL develops in Cbfa2t3 heterozygotes with a significantly longer latency, suggesting that PRDM14-associated T-ALL is sensitive to Cbfa2t3 levels. Our study highlights how an oncogenic protein uses a native protein in progenitor cells to initiate leukemia, providing insight into PRDM14-driven oncogenesis in other cell types. IMPLICATIONS: The pluripotency regulator PRDM14 requires the master hematopoietic regulator CBFA2T3 to initiate leukemia in progenitor cells, demonstrating an oncogenic role for CBFA2T3 and providing an avenue for targeting cancer-initiating cells.

Co-option of the PRDM14–CBFA2T complex from motor neurons to pluripotent cells during vertebrate evolution

Gene regulatory networks underlying cellular pluripotency are controlled by a core circuitry of transcription factors in mammals, including POU5F1. However, the evolutionary origin and transformation of pluripotency-related transcriptional networks have not been elucidated in deuterostomes. PR domain-containing protein 14 (PRDM14) is specifically expressed in pluripotent cells and germ cells, and is required for establishing embryonic stem cells (ESCs) and primordial germ cells in mice. Here, we compared the functions and expression patterns of PRDM14 orthologues within deuterostomes. Amphioxus PRDM14 and zebrafish PRDM14, but not sea urchin PRDM14, compensated for mouse PRDM14 function in maintaining mouse ESC pluripotency. Interestingly, sea urchin PRDM14 together with sea urchin CBFA2T, an essential partner of PRDM14 in mouse ESCs, complemented the self-renewal defect in mouse Prdm14 KO ESCs. Contrary to the Prdm14 expression pattern in mouse embryos, Prdm14 was expressed in motor neurons of amphioxus embryos, as observed in zebrafish embryos. Thus, Prdm14 expression in motor neurons was conserved in non-tetrapod deuterostomes and the co-option of the PRDM14-CBFA2T complex from motor neurons into pluripotent cells may have maintained the transcriptional network for pluripotency during vertebrate evolution.This article has an associated 'The people behind the papers' interview.

PRDM14: A Potential Target for Cancer Therapy.

PRDM14 belongs to the PR domain-containing (PRDM) family. Although a precise understanding focused on the function of PRDM14 to maintain stemness and pluripotency in embryonic stem cells via epigenetic mechanisms, growing experimental evidence has been linked PRDM14 to human cancers. In adults, PRDM14 has low expression in human tissues. Aberrant PRDM14 expression is connected with various malignant histological types and solid cancers, where PRDM14 can act as a driver of oncogenic processes. Overexpression of RPDM14 enhanced cancer cells growth and reduced cancer cells sensitive to chemotherapeutic agents. Reducing the expression of PRDM14 in cancer cells can enhance the therapeutic sensitivity of drugs to cancer cells, suggesting that aberrant PRDM14 may have a carcinogenic characteristic in tumor therapy and as a new molecular target. This review summarizes the structure and oncogenic properties of PRDM14 in different malignancies and suggests that PRDM14 may be a potential therapeutic molecular target for tumor treatment.

PRDM14 is overexpressed in chronic pancreatitis prior to pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer that is typically diagnosed at a later stage with metastases and is difficult to treat. Therefore, investigating the mechanism of PDAC initiation is important to aid early‐stage cancer detection. PRDM14 is a transcription factor that maintains pluripotency in embryonic stem cells and is overexpressed in several cancers. We previously reported that PRDM14 is overexpressed and regulates cancer stem‐like phenotypes in PDAC, and herein, we assess whether PRDM14 expression increases prior to tumorigenesis. Through immunohistochemistry analyses of clinical tissues, we detected PRDM14‐positive cells in precursor pancreatic intraepithelial neoplasia and chronic pancreatitis, which is a risk factor for PDAC, lesions. PRDM14 staining in chronic pancreatitis was as high as that in PDAC and cancer adjacent tissues. We induced pancreatitis in mouse models by cerulein injection, and observed that PRDM14 expression increased in chronic pancreatitis models but not in control or acute pancreatitis mice. Moreover, cerulein treatment increased PRDM14 expression in PK‐1 and AsPC‐1 pancreatic cancer cell lines. Our results suggest that inflammation increases the expression of PRDM14, which regulates cancer stem‐like phenotypes, and this occurs prior to PDAC initiation and progression.

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but naïve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78.

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem‐like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple‐negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates—glucose‐regulated protein 78 (GRP78) and heat shock protein 90‐α (HSP90α)—were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA‐MB231). Surface plasmon resonance analysis using GST‐PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C‐terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase‐based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem‐like CD24− CD44+ and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation.

Sex chromosomes drive gene expression and regulatory dimorphisms in mouse embryonic stem cells

BackgroundPre-implantation embryos exhibit sexual dimorphisms in both primates and rodents. To determine whether these differences reflected sex-biased expression patterns, we generated transcriptome profiles for six 40,XX, six 40,XY, and two 39,X mouse embryonic stem (ES) cells by RNA sequencing.ResultsWe found hundreds of coding and non-coding RNAs that were differentially expressed between male and female cells. Surprisingly, the majority of these were autosomal and included RNA encoding transcription and epigenetic and chromatin remodeling factors. We showed differential Prdm14-responsive enhancer activity in male and female cells, correlating with the sex-specific levels of Prdm14 expression. This is the first time sex-specific enhancer activity in ES cells has been reported. Evaluation of X-linked gene expression patterns between our XX and XY lines revealed four distinct categories: (1) genes showing 2-fold greater expression in the female cells; (2) a set of genes with expression levels well above 2-fold in female cells; (3) genes with equivalent RNA levels in male and female cells; and strikingly, (4) a small number of genes with higher expression in the XY lines. Further evaluation of autosomal gene expression revealed differential expression of imprinted loci, despite appropriate parent-of-origin patterns. The 39,X lines aligned closely with the XY cells and provided insights into potential regulation of genes associated with Turner syndrome in humans. Moreover, inclusion of the 39,X lines permitted three-way comparisons, delineating X and Y chromosome-dependent patterns.ConclusionsOverall, our results support the role of the sex chromosomes in establishing sex-specific networks early in embryonic development and provide insights into effects of sex chromosome aneuploidies originating at those stages.

THAP1: Role in Mouse Embryonic Stem Cell Survival and Differentiation.

SummaryTHAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a ubiquitously expressed member of a family of transcription factors with highly conserved DNA-binding and protein-interacting regions. Mutations in THAP1 cause dystonia, DYT6, a neurologic movement disorder. THAP1 downstream targets and the mechanism via which it causes dystonia are largely unknown. Here, we show that wild-type THAP1 regulates embryonic stem cell (ESC) potential, survival, and proliferation. Our findings identify THAP1 as an essential factor underlying mouse ESC survival and to some extent, differentiation, particularly neuroectodermal. Loss of THAP1 or replacement with a disease-causing mutation results in an enhanced rate of cell death, prolongs Nanog, Prdm14, and/or Rex1 expression upon differentiation, and results in failure to upregulate ectodermal genes. ChIP-Seq reveals that these activities are likely due in part to indirect regulation of gene expression.

Inhibition of PRDM14 expression in pancreatic cancer suppresses cancer stem-like properties and liver metastasis in mice.

Pancreatic cancer is one of the most lethal types of cancer, with aggressive properties characterized by metastasis, recurrence and drug resistance. Cancer stem cells are considered to be responsible for these properties. PRDM14, a transcriptional regulator that maintains pluripotency in embryonic stem cells, is overexpressed in some cancers. Here, we assessed PRDM14 expression and the effects of PRDM14 knockdown on cancer stem-like phenotypes in pancreatic cancer. We observed that PRDM14 protein was overexpressed in pancreatic cancer tissues compared with normal pancreatic tissues. Using lentiviral shRNA-transduced pancreatic cancer cells, we found that PRDM14 knockdown decreased sphere formation, number of side population and cell surface marker-positive cells and subcutaneous xenograft tumors and liver metastasis in mice. This was accompanied by upregulation of some microRNAs (miRNAs), including miR-125a-3p. miR-125a-3p, a tumor suppressor that is down-regulated in pancreatic cancer, has been suggested to regulate the expression of the Src-family kinase, Fyn. In PRDM14-knockdown cells, Fyn was expressed at lower levels and downstream proteins were less activated. These changes were considered to cause suppression of the above cancer phenotypes. In addition, we used small interfering RNA (siRNA)-based therapy targeting PRDM14 in a mouse model of liver metastasis induced using MIA-PaCa2 cells, and this treatment significantly decreased metastasis and in vitro migration. Taken together, these results suggest that targeting the overexpression of PRDM14 suppresses cancer stem-like phenotypes, including liver metastasis, via miRNA regulation and siRNA-based therapy targeting it shows promise as a treatment for patients with pancreatic cancer.

2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms.

SummaryMouse embryonic stem cells (ESCs) are maintained in serum with leukemia inhibitory factor (LIF) to maintain self-renewal and pluripotency. Recently, a 2i culture method was reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintain ESCs in a naive ground state. How 2i maintains a ground state of ESCs remains elusive. Here we show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine (5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintain a naive ground state through JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation.

Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling.

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

Histone demethylation maintains Prdm14 and Tsix expression and represses xIst in embryonic stem cells.

Epigenetic reprogramming is exemplified by the remarkable changes observed in cellular differentiation and X-chromosome inactivation (XCI) in mammalian female cells. Histone 3 lysine 27 trimethylation (H3K27me3) is a modification that suppresses gene expression in multiple contexts including embryonic stem cells (ESCs) and decorates the entire inactive X-chromosome. The conversion of female somatic cells to induced pluripotency is accompanied by X-chromosome reactivation (XCR) and H3K27me3 erasure. Here, we show that the H3K27-specific demethylase Utx regulates the expression of the master regulators for XCI and XCR: Prdm14, Tsix, and Xist. Female ESC transcriptome analysis using a small molecule inhibitor for H3K27 demethylases, GSK-J4, identifies novel targets of H3K27 demethylation. Consistent with a recent report that GSK-J4 can inhibit other histone demethylase, we found that elevated H3K4me3 levels are associated with increased gene expression including Xist. Our data suggest multiple regulatory mechanisms for XCI via histone demethylation.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile.

Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3–6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.

PRDM14 promotes active DNA demethylation through the Ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

High expression of PRDM14 correlates with cell differentiation and is a novel prognostic marker in resected non-small cell lung cancer

PR (PRDI-BF1 and RIZ) domain containing proteins (PRDM) have been indicated to play important roles in several human cancers. The objectives of this study were to determine the frequency and prognostic significance of PRDM1 and PRDM14 expression in a cohort of surgically resected non-small cell lung cancer (NSCLC) patients. Immunohistochemistry and Western blotting was used to detect the expression status of PRDM1 in primary tumors and PRDM14 for both primary lung cancers and matched lymph node metastases. Univariate and multivariate analysis were performed to determine the association between PRDM expression and prognosis. PRDM1 expression was observed in all NSCLC patients' tumor samples. PRDM14 was found to be increased expression in 35.65 % cases (46/129) for primary tumors and 39.68 % cases (25/63) for paired metastatic lymph nodes. Increased expression of PRDM14 correlated with differentiation of tumor cells significantly (P = 0.008). Western blotting analysis verified that PRDM14 associated with cell differentiation in NSCLC. The overall survival rates of patients with high PRDM14 expression and low PRDM14 expression were 41.30 and 65.06 %, respectively (hazard ratio: 3.051, 95 % CI: 1.752, 5.312, P < 0.0001). The progression-free survival rates were 34.78 % for patients in the high expression group and 59.03 % for patients in the low OLC1 expression group (hazard ratio: 2.775, 95 % CI: 1.648, 4.675, P < 0.0001). Thus, our study showed that increased expression of PRDM14 correlated with cell differentiation of NSCLC cells. PRDM14 was a potential biomarker for predicting unfavorable prognosis in NSCLC.