DPPA4 increases aggressiveness of pituitary neuroendocrine tumors by enhancing cell stemness.

BACKGROUND: Pituitary neuroendocrine tumors, PitNETs, are often aggressive and precipitate in distant metastases that are refractory to current therapies. However, the molecular mechanism in PitNETs' aggressiveness is not well understood. Developmental pluripotency-associated 4 (DPPA4) is known as a stem cell regulatory gene and overexpressed in certain cancers, but its function in the context of PitNETs' aggressiveness is not known. METHODS: We employed both rat and human models of PitNETs. In the rat pituitary tumor model (RPT), we used prenatal-alcohol-exposed (PAE) female Fischer rats which developed aggressive PitNETs following estrogen treatment, while in the human pituitary tumor (HPT) model, we used aggressively proliferative cells from pituitary tumors of patients undergone surgery. Various molecular, cellular, and epigenetic techniques were used to determine the role of DPPA4 in PitNETs' aggressiveness. RESULTS: We show that DPPA4 is overexpressed in association with increased cell stemness factors in aggressive PitNETs of PAE rats and of human patients. Gene-editing experiments demonstrate that DPPA4 increases the expression of cell stemness and tumor aggressiveness genes and promotes proliferation, colonization, migration, and tumorigenic potential of PitNET cells. ChIP assays and receptor antagonism studies reveal that DPPA4 binds to canonical WINTs promoters and increases directly or indirectly the Wnt/ß-catenin control of cell stemness, tumor growth, and aggressiveness of PitNETs. Epigenetic studies show involvement of histone methyltransferase in alcohol activation of DPPA4. CONCLUSIONS: These findings support a role of DPPA4 in tumor stemness and aggressiveness and provide a preclinical rationale for modulating this stemness regulator for the treatment of PitNETs.

DPPA2/4 Promote the Pluripotency and Proliferation of Bovine Extended Pluripotent Stem Cells by Upregulating the PI3K/AKT/GSK3ß/ß-Catenin Signaling Pathway.

Developmental pluripotency-associated 2 (DPPA2) and DPPA4 are crucial transcription factors involved in maintaining pluripotency in humans and mice. However, the role of DPPA2/4 in bovine extended pluripotent stem cells (bEPSCs) has not been investigated. In this study, a subset of bEPSC-related differentially expressed genes (DEGs), including DPPA2 and DPPA4, was identified based on multiomics data (ATAC-seq and RNA-seq). Subsequent investigations revealed that double overexpression of DPPA2/4 facilitates the reprogramming of bovine fetal fibroblasts (BFFs) into bEPSCs, whereas knockout of DPPA2/4 in BFFs leads to inefficient reprogramming. DPPA2/4 overexpression and knockdown experiments revealed that the pluripotency and proliferation capability of bEPSCs were maintained by promoting the transition from the G1 phase to the S phase of the cell cycle. By activating the PI3K/AKT/GSK3ß/ß-catenin pathway in bEPSCs, DPPA2/4 can increase the nuclear accumulation of ß-catenin, which further upregulates lymphoid enhancer binding factor 1 (LEF1) transcription factor activity. Moreover, DPPA2/4 can also regulate the expression of LEF1 by directly binding to its promoter region. Overall, our results demonstrate that DPPA2/4 promote the reprogramming of BFFs into bEPSCs while also maintaining the pluripotency and proliferation capability of bEPSCs by regulating the PI3K/AKT/GSK3ß/ß-catenin pathway and subsequently activating LEF1. These findings expand our understanding of the gene regulatory network involved in bEPSC pluripotency.

Chromatin modifier developmental pluripotency associated factor 4 (DPPA4) is a candidate gene for alcohol-induced developmental disorders.

BACKGROUND: Prenatal alcohol exposure (PAE) affects embryonic development, causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neuronal disorders and birth defects. We hypothesize that early alcohol-induced epigenetic changes disrupt the accurate developmental programming of embryo and consequently cause the complex phenotype of developmental disorders. To explore the etiology of FASD, we collected unique biological samples of 80 severely alcohol-exposed and 100 control newborns at birth. METHODS: We performed genome-wide DNA methylation (DNAm) and gene expression analyses of placentas by using microarrays (EPIC, Illumina) and mRNA sequencing, respectively. To test the manifestation of observed PAE-associated DNAm changes in embryonic tissues as well as potential biomarkers for PAE, we examined if the changes can be detected also in white blood cells or buccal epithelial cells of the same newborns by EpiTYPER. To explore the early effects of alcohol on extraembryonic placental tissue, we selected 27 newborns whose mothers had consumed alcohol up to gestational week 7 at maximum to the separate analyses. Furthermore, to explore the effects of early alcohol exposure on embryonic cells, human embryonic stem cells (hESCs) as well as hESCs during differentiation into endodermal, mesodermal, and ectodermal cells were exposed to alcohol in vitro. RESULTS: DPPA4, FOXP2, and TACR3 with significantly decreased DNAm were discovered-particularly the regulatory region of DPPA4 in the early alcohol-exposed placentas. When hESCs were exposed to alcohol in vitro, significantly altered regulation of DPPA2, a closely linked heterodimer of DPPA4, was observed. While the regulatory region of DPPA4 was unmethylated in both control and alcohol-exposed hESCs, alcohol-induced decreased DNAm similar to placenta was seen in in vitro differentiated mesodermal and ectodermal cells. Furthermore, common genes with alcohol-associated DNAm changes in placenta and hESCs were linked exclusively to the neurodevelopmental pathways in the enrichment analysis, which emphasizes the value of placental tissue when analyzing the effects of prenatal environment on human development. CONCLUSIONS: Our study shows the effects of early alcohol exposure on human embryonic and extraembryonic cells, introduces candidate genes for alcohol-induced developmental disorders, and reveals potential biomarkers for prenatal alcohol exposure.

Dynamic cytosolic foci of DPPA4 in human pluripotent stem cells.

DPPA4 is essential for the pluripotent stem cell state, yet its function is poorly understood. DPPA4 is localized in the nucleus, where it is associated with active chromatin. We now report that it is also present in the cytosol, where it appears as diffused clouds, distinct foci and sometimes as spaghetti-like structures. This cytosolic localization is dynamic and DPPA4 shuttles between the cytosol and the nucleus. Its presence is almost abolished from the nucleus upon differentiation. Co-immunoprecipitation studies highlighted novel protein interactors, many of which are also found in the cytosol and are implicated in mRNA processing and RNA and protein transport between the cytosol and the nucleus. Finally, the depletion of DPPA4 resulted in cytosolic accumulation of vesicles. The cytosolic presence of DPPA4 highlights unexplored research directions that could significantly advance the understanding of DPPA4 in pluripotent stem cells and in cancer.

Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival.

Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

DPPA2, DPPA4, and other DPPA factor epigenomic functions in cell fate and cancer.

Many gene networks are shared between pluripotent stem cells and cancer; a concept exemplified by several DPPA factors such as DPPA2 and DPPA4, which are highly and selectively expressed in stem cells but also found to be reactivated in cancer. Despite their striking expression pattern, for many years the function of DPPA2 and DPPA4 remained a mystery; knockout of Dppa2 and Dppa4 did not affect pluripotency, but caused lung and skeletal defects late in development, long after Dppa2 and Dppa4 expression had been turned off. A number of recent papers have further clarified and defined the roles of these important factors, identifying roles in priming the chromatin and maintaining developmental competency through regulating both H3K4me3 and H3K27me3 at bivalent chromatin domains, and acting to remodel chromatin and facilitate reprogramming of somatic cells to induced pluripotency. These findings highlight an important regulatory role for DPPA2 and DPPA4 at the transitional boundary between pluripotency and differentiation and may have relevance to the functions of DPPA2 and 4 in the context of cancer cells as well.

Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

DPPA2 and DPPA4 are necessary to establish a 2C-like state in mouse embryonic stem cells.

After fertilization of the transcriptionally silent oocyte, expression from both parental chromosomes is launched through zygotic genome activation (ZGA), occurring in the mouse at the 2-cell (2C) stage. Among the first elements to be transcribed are the Dux gene, the product of which induces a wide array of ZGA genes, and a subset of evolutionary recent LINE-1 retrotransposons that regulate chromatin accessibility in the early embryo. The maternally inherited factors that activate Dux and LINE-1 transcription have so far remained unknown. Mouse embryonic stem cells (mESCs) recapitulate some aspects of ZGA in culture, owing to their ability to cycle through a 2C-like stage when Dux, its target genes, and LINE-1 integrants are expressed. Here, we identify the paralog proteins DPPA2 and DPPA4 as necessary for the activation of Dux and LINE-1 expression in mESCs. Since their encoding RNAs are maternally transmitted to the zygote, it is likely that these factors are important upstream mediators of murine ZGA.

Dppa2 and Dppa4 directly regulate the Dux-driven zygotic transcriptional program.

The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.

Genomic functions of developmental pluripotency associated factor 4 (Dppa4) in pluripotent stem cells and cancer.

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC), embryonal carcinoma cells, and 3T3 fibroblasts ectopically expressing Dppa4. We find a core set of Dppa4 binding sites shared across cell types, and also a substantial number of sites unique to each cell type. Across cell types Dppa4 shows a preference for binding to regions with active chromatin signatures, and can influence chromatin modifications at target genes. In 3T3 fibroblasts with enforced Dppa4 expression, Dppa4 represses the cell cycle inhibitor Cdkn2c and activates Ets family transcription factor Etv4, leading to alterations in the cell cycle that likely contribute to the oncogenic phenotype. Dppa4 also directly regulates Etv4 in mESC but represses it in this context, and binds with Oct4 to a set of shared targets that are largely independent of Sox2 and Nanog, indicating that Dppa4 functions independently of the core pluripotency network in stem cells. Together these data provide novel insights into Dppa4 function in both pluripotent and oncogenic contexts.

ERBB3-Binding Protein 1 (EBP1) Is a Novel Developmental Pluripotency-Associated-4 (DPPA4) Cofactor in Human Pluripotent Cells.

Developmental Pluripotency-Associated-4 (DPPA4) is one of the few core pluripotency genes lacking clearly defined molecular and cellular functions. Here, we used a proteomics screening approach of human embryonic stem cell (hESC) nuclear extract to determine DPPA4 molecular functions through identification of novel cofactors. Unexpectedly, the signaling molecule ERBB3-binding protein 1 (EBP1) was the strongest candidate binding partner for DPPA4 in hESC. EBP1 is a growth factor signaling mediator present in two isoforms, p48 and p42. The two isoforms generally have opposing functions, however their roles in pluripotent cells have not been established. We found that DPPA4 preferentially binds p48 in pluripotent and NTERA-2 cells, but this interaction is largely absent in non-pluripotent cells and is reduced with differentiation. The DPPA4-EBP1 interaction is mediated at least in part in DPPA4 by the highly conserved SAF-A/B, Acinus and PIAS (SAP) domain. Functionally, we found that DPPA4 transcriptional repressive function in reporter assays is significantly increased by specific p48 knockdown, an effect that was abolished with an interaction-deficient DPPA4 ΔSAP mutant. Thus, DPPA4 and EBP1 may cooperate in transcriptional functions through their physical association in a pluripotent cell specific context. Our study identifies EBP1 as a novel pluripotency cofactor and provides insight into potential mechanisms used by DPPA4 in regulating pluripotency through its association with EBP1. Stem Cells 2018;36:671-682.

Compartmentalization of HP1 Proteins in Pluripotency Acquisition and Maintenance.

The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1ß knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency.

Copy number increase of oncoprotein CIP2A is associated with poor patient survival in human head and neck squamous cell carcinoma.

BACKGROUND: CIP2A, an inhibitor of PP2A tumour suppressor function, is a widely overexpressed biomarker of aggressive disease and poor therapy response in multiple human cancer types. METHODS: CIP2A and DPPA4 copy number alterations and expression were analysed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in different cell lines and a tissue microarray of 52 HNSCC patients. Results were correlated with patient survival and other clinicopathological data. RESULTS: CIP2A and DPPA4 copy number increase occurred at a relatively high frequency in human HNSCC patient samples. CIP2A but not DPPA4 FISH status was significantly associated with patient survival. CIP2A detection by combining IHC with FISH yielded superior resolution in the prognostication of HNSCC. CONCLUSIONS: CIP2A copy number increase is associated with poor patient survival in human HNSCC. We suggest that the reliability and prognostic value of CIP2A detection can be improved by performing FISH analysis to CIP2A IHC positive tumours.

Identification of DPPA4 and DPPA2 as a novel family of pluripotency-related oncogenes.

In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated four) that encodes a DNA binding SAP domain-containing protein. DPPA4 has not been previously identified as an oncogene but is highly expressed in embryonal carcinomas, pluripotent germ cell tumors, and other cancers. DPPA4 is also mutated in some cancers. In direct transformation assays, we validated that DPPA4 is an oncogene in both mouse 3T3 cells and immortalized human dermal fibroblasts. Overexpression of DPPA4 generates oncogenic foci (sarcoma cells) and causes anchorage-independent growth. The in vitro transformed cells also give rise to tumors in immunodeficient mice. Furthermore, functional analyses indicate that both the DNA-binding SAP domain and the histone-binding C-terminal domain are critical for the oncogenic transformation activity of DPPA4. Downregulation of DPPA4 in E14 mouse embryonic stem cells and P19 mouse embryonic carcinoma cells causes decreased cell proliferation in each case. In addition, DPPA4 overexpression induces cell proliferation through genes related to regulation of G1/S transition. Interestingly, we observed similar findings for family member DPPA2. Thus, we have identified a new family of pluripotency-related oncogenes consisting of DPPA2 and DPPA4. Our findings have important implications for stem cell biology and tumorigenesis.