Mammalian Embryonic Stem Cells Research Articles

Abstract 3526: GAIN-BRCA: A graphical explainable AI-net framework for breast cancer subtype classification using multiomics data

Abstract Higher standards for precise distinction and classification among the four breast cancer subtypes, i.e., Luminal A. Luminal B, HER-2-enriched, and Basal, are essential to delivering customized treatment in precision oncology. The effective integration of readily accessible multi-omics datasets from the same patient may improve the accuracy of subtype prediction algorithms and help decipher reliable molecular features for the prediction and prognosis of breast cancer. Previous models for predicting breast cancer subtypes relied mostly on concatenation and autoencoder methods but rarely utilized graph-based integration to leverage the biological associations among omics datatypes. To overcome these limitations, we developed a graph-based multi-omics integration method using features from mRNA, DNA methylation, and miRNA data and synthesized features from their interactions. GAIN-BRCA computes weightage scores from miRNA-mRNA and DNA methylation-mRNA interactions to derive a new transformed feature vector without compromising biological information. The neural network architecture was designed and trained on the transformed features to classify breast cancer subtypes. Neural network prediction accuracies were compared using concatenated, autoencoder, and GAIN-BRCA-based integrated datasets. GAIN-BRCA outperforms the existing similar models MOGONET (Acc: 0.72) and moBRCA-net (Acc: 0.86) with an accuracy of 0.91. These transformed features were then processed using SHAP, and the top 357 features were selected for functional characterization and pathway analysis. We identified specific pathways and genes related to each breast cancer subtype separately. The most enriched pathways found in each subtype are ribonucleotide reductase signaling and glucocorticoid signaling pathways in luminal A, PFKB4 signaling pathway in luminal B, adipogenesis and DNA methylation and transcriptional signaling in HER2+, and the role of OCT4 in mammalian embryonic stem cell pluripotency in basal. Similarly, the most discriminated gene features are BRAF, ESR1, KRAS, MAPK1, SMARCE1, PPP3CA, and TGFBR2 in luminal A, TGFB3 and NCOA3 in luminal B, HNF4A, SIN3A, and SOX9 in HER2+, and POU5F1 and FOXA2 in basal subtypes. The GAIN-BRCA framework, combined with SHAP, weighs independent omics as cross-disciplinary rather than multidisciplinary and presents a distinctive picture for each subtype. As a result, the framework presents the importance of features about the corresponding subtype, which might lead to a greater understanding of the molecular basis of breast cancer subtypes. Citation Format: Jai Chand Patel, Sushil Kumar Shakyawar, Sahil Sethi, Chittibabu Guda. GAIN-BRCA: A graphical explainable AI-net framework for breast cancer subtype classification using multiomics data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3526.

Roles and Regulation of H3K4 Methylation During Mammalian Early Embryogenesis and Embryonic Stem Cell Differentiation.

From generation of germ cells, fertilization, and throughout early mammalian embryonic development, the chromatin undergoes significant alterations to enable precise regulation of gene expression and genome use. Methylation of histone 3 lysine 4 (H3K4) correlates with active regions of the genome, and it has emerged as a dynamic mark throughout this timeline. The pattern and the level of H3K4 methylation are regulated by methyltransferases and demethylases. These enzymes, as well as their protein partners, play important roles in early embryonic development and show phenotypes in embryonic stem cell self-renewal and differentiation. The various roles of H3K4 methylation are interpreted by dedicated chromatin reader proteins, linking this modification to broader molecular and cellular phenotypes. In this review, we discuss the regulation of different levels of H3K4 methylation, their distinct accumulation pattern, and downstream molecular roles with an early embryogenesis perspective.

Micelle-like clusters in phase-separated Nanog condensates: A molecular simulation study.

The phase separation model for transcription suggests that transcription factors (TFs), coactivators, and RNA polymerases form biomolecular condensates around active gene loci and regulate transcription. However, the structural details of condensates remain elusive. In this study, for Nanog, a master TF in mammalian embryonic stem cells known to form protein condensates in vitro, we examined protein structures in the condensates using residue-level coarse-grained molecular simulations. Human Nanog formed micelle-like clusters in the condensate. In the micelle-like cluster, the C-terminal disordered domains, including the tryptophan repeat (WR) regions, interacted with each other near the cluster center primarily via hydrophobic interaction. In contrast, hydrophilic disordered N-terminal and DNA-binding domains were exposed on the surface of the clusters. Electrostatic attractions of these surface residues were responsible for bridging multiple micelle-like structures in the condensate. The micelle-like structure and condensate were dynamic and liquid-like. Mutation of tryptophan residues in the WR region which was implicated to be important for a Nanog function resulted in dissolution of the Nanog condensate. Finally, to examine the impact of Nanog cluster to DNA, we added DNA fragments to the Nanog condensate. Nanog DNA-binding domains exposed to the surface of the micelle-like cluster could recruit more than one DNA fragments, making DNA-DNA distance shorter.

The CRHR1/CREB/REST signaling cascade regulates mammalian embryonic neural stem cell properties.

Growing evidence suggests that the corticotropin-releasing hormone (CRH) signaling pathway, mainly known as a critical initiator of humoral stress responses, has a role in normal neuronal physiology. However, despite the evidence of CRH receptor (CRHR) expression in the embryonic ventricular zone, the exact functions of CRH signaling in embryonic brain development have not yet been fully determined. In this study, we show that CRHR1 is required for the maintenance of neural stem cell properties, as assessed by invitro neurosphere assays and cell distribution in the embryonic cortical layers following in utero electroporation. Identifying the underlying molecular mechanisms of CRHR1 action, we find that CRHR1 functions are accomplished through the increasing expression of the master transcription factor REST. Furthermore, luciferase reporter and chromatin immunoprecipitation assays reveal that CRHR1-induced CREB activity is responsible for increased REST expression at the transcriptional level. Taken together, these findings indicate that the CRHR1/CREB/REST signaling cascade plays an important role downstream of CRH in the regulation of neural stem cells during embryonic brain development.

Cellular senescence, rejuvenation and potential immortality.

Ageing, death, and potential immortality lie at the heart of biology, but two seemingly incompatible paradigms coexist in different research communities and have done since the nineteenth century. The universal senescence paradigm sees senescence as inevitable in all cells. Damage accumulates. The potential immortality paradigm sees some cells as potentially immortal, especially unicellular organisms, germ cells and cancerous cells. Recent research with animal cells, yeasts and bacteria show that damaged cell constituents do in fact build up, but can be diluted by growth and cell division, especially by asymmetric cell division. By contrast, mammalian embryonic stem cells and many cancerous and ‘immortalized’ cell lines divide symmetrically, and yet replicate indefinitely. How do they acquire their potential immortality? I suggest they are rejuvenated by excreting damaged cell constituents in extracellular vesicles. If so, our understanding of cellular senescence, rejuvenation and potential immortality could be brought together in a new synthesis, which I call the cellular rejuvenation hypothesis: damaged cell constituents build up in all cells, but cells can be rejuvenated either by growth and cell division or, in ‘immortal’ cell lines, by excreting damaged cell constituents. In electronic supplementary material, appendix, I outline nine ways in which this hypothesis could be tested.

Statistical estimates of multiple transcription factors binding in the model plant genomes based on ChIP-seq data.

The development of high-throughput genomic sequencing coupled with chromatin immunoprecipitation technologies allows studying the binding sites of the protein transcription factors (TF) in the genome scale. The growth of data volume on the experimentally determined binding sites raises qualitatively new problems for the analysis of gene expression regulation, prediction of transcription factors target genes, and regulatory gene networks reconstruction. Genome regulation remains an insufficiently studied though plants have complex molecular regulatory mechanisms of gene expression and response to environmental stresses. It is important to develop new software tools for the analysis of the TF binding sites location and their clustering in the plant genomes, visualization, and the following statistical estimates. This study presents application of the analysis of multiple TF binding profiles in three evolutionarily distant model plant organisms. The construction and analysis of non-random ChIP-seq binding clusters of the different TFs in mammalian embryonic stem cells were discussed earlier using similar bioinformatics approaches. Such clusters of TF binding sites may indicate the gene regulatory regions, enhancers and gene transcription regulatory hubs. It can be used for analysis of the gene promoters as well as a background for transcription networks reconstruction. We discuss the statistical estimates of the TF binding sites clusters in the model plant genomes. The distributions of the number of different TFs per binding cluster follow same power law distribution for all the genomes studied. The binding clusters in Arabidopsis thaliana genome were discussed here in detail.

Transposable Element Dynamics and Regulation during Zygotic Genome Activation in Mammalian Embryos and Embryonic Stem Cell Model Systems.

Transposable elements (TEs) are mobile genetic sequences capable of duplicating and reintegrating at new regions within the genome. A growing body of evidence has demonstrated that these elements play important roles in host genome evolution, despite being traditionally viewed as parasitic elements. To prevent ectopic activation of TE transposition and transcription, they are epigenetically silenced in most somatic tissues. Intriguingly, a specific class of TEs—retrotransposons—is transiently expressed at discrete phases during mammalian development and has been linked to the establishment of totipotency during zygotic genome activation (ZGA). While mechanisms controlling TE regulation in somatic tissues have been extensively studied, the significance underlying the unique transcriptional reactivation of retrotransposons during ZGA is only beginning to be uncovered. In this review, we summarize the expression dynamics of key retrotransposons during ZGA, focusing on findings from in vivo totipotent embryos and in vitro totipotent-like embryonic stem cells (ESCs). We then dissect the functions of retrotransposons and discuss how their transcriptional activities are finetuned during early stages of mammalian development.

Rapid fabrication of hydrogel micropatterns by projection stereolithography for studying self-organized developmental patterning.

Self-organized patterning of mammalian embryonic stem cells on micropatterned surfaces has previously been established as an in vitro platform for early mammalian developmental studies, complimentary to in vivo studies. Traditional micropatterning methods, such as micro-contact printing (μCP), involve relatively complicated fabrication procedures, which restricts widespread adoption by biologists. Here, we demonstrate a rapid method of micropatterning by printing hydrogel micro-features onto a glass-bottomed culture vessel. The micro-features are printed using a projection stereolithography bioprinter yielding hydrogel structures that geometrically restrict the attachment of cells or proteins. Compared to traditional and physical photomasks, a digitally tunable virtual photomask is used in the projector to generate blue light patterns that enable rapid iteration with minimal cost and effort. We show that a protocol that makes use of this method together with LN521 coating, an extracellular matrix coating, creates a surface suitable for human embryonic stem cell (hESC) attachment and growth with minimal non-specific adhesion. We further demonstrate that self-patterning of hESCs following previously published gastrulation and ectodermal induction protocols achieves results comparable with those obtained with commercially available plates.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

97 Function of species-specific OCT4 reporter system during porcine embryo development

The present study examined the activity and function of pig OCT4 (POU5F1) enhancer in porcine early embryo development. OCT4 is one of the master regulators for the pluripotency of early mammalian embryonic development and embryonic stem cells. It has two regulatory elements: distal enhancer (DE) and proximal enhancer (PE) in the upstream regulatory region. They are activated under different conditions such as naïve and primed. It is known that two enhancers are activated to produce OCT4 simultaneously or sequentially depending on the state of pluripotency. Many porcine OCT4 upstream region-based reporter systems have been reported because this is a necessary part of studying porcine-specific pluripotency. However, the porcine-specific OCT4 reporter system has never been transfected and functionally tested during porcine early embryo development. We performed functional tests of the previously established porcine-specific OCT4 reporter system in the early embryo development stage. Porcine embryos were micro-injected with the pOCT4-ΔPE-eGFP (DE-GFP) containing a distal enhancer and core promoter and pOCT4-ΔDE-DsRed2 (PE-RFP) containing a proximal enhancer and core promoter. They were cultured in PZM-3 at 39°C in a humidified atmosphere, 5% CO2, and 5% O2 for 168h. Fifty of the 100 embryos were injected with OCT4 reporter system as treatment groups and the other 50 were not injected (control groups). We analysed mRNA and protein expression of GFP and RFP using quantitative real-time PCR and confocal microscopy by developmental stages. The introduced reporter system could function beginning with the 4-cell stage. The expression of GFP and RFP was observed simultaneously in the early embryonic development stage up to blastocyst, indicating that porcine OCT4 was produced by distal and proximal enhancers, unlike mouse Oct4 expression, which was only controlled by the distal enhancer during early embryo development. Therefore, the mechanisms and functions of distal and proximal enhancers of porcine OCT4 were different from those of the mouse. This is similar to results of the previous experiment using porcine-induced pluripotent stem cells, which suggest that porcine OCT4 is expressed by two enhancers and in a stage-specific manner during reprogramming and that pigs do not use only one enhancer in pluripotent states, but two enhancers at the same time, and there is only a difference in degree. This work showed that the porcine OCT4 reporter system enables non-destructive analysis during early embryo development and it could be applied to study species-specific pluripotency and to help the establishment of naïve embryonic stem cells in the pig. This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).

Neural induction: Historical views and application to pluripotent stem cells.

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state-of-the-art stem cell research.

A Genetic Screen Identifies Etl4-Deficiency Capable of Stabilizing the Haploidy in Embryonic Stem Cells.

SummaryMammalian haploid embryonic stem cells (haESCs) hold great promise for functional genetic studies and forward screening. However, all established haploid cells are prone to spontaneous diploidization during long-term culture, rendering application challenging. Here, we report a genome-wide loss-of-function screening that identified gene mutations that could significantly reduce the rate of self-diploidization in haESCs. We further demonstrated that CRISPR/Cas9-mediated Etl4 knockout (KO) stabilizes the haploid state in different haESC lines. More interestingly, Etl4 deficiency increases mitochondrial oxidative phosphorylation (OXPHOS) capacity and decreases glycolysis in haESCs. Mimicking this effect by regulating the energy metabolism with drugs decreased the rate of self-diploidization. Collectively, our study identified Etl4 as a novel haploidy-related factor linked to an energy metabolism transition occurring during self-diploidization of haESCs.

Developmental remodelling of non-CG methylation at satellite DNA repeats.

In vertebrates, DNA methylation predominantly occurs at CG dinucleotides however, widespread non-CG methylation (mCH) has been reported in mammalian embryonic stem cells and in the brain. In mammals, mCH is found at CAC trinucleotides in the nervous system, where it is associated with transcriptional repression, and at CAG trinucleotides in embryonic stem cells, where it positively correlates with transcription. Moreover, CAC methylation appears to be a conserved feature of adult vertebrate brains. Unlike any of those methylation signatures, here we describe a novel form of mCH that occurs in the TGCT context within zebrafish mosaic satellite repeats. TGCT methylation is inherited from both male and female gametes, remodelled during mid-blastula transition, and re-established during gastrulation in all embryonic layers. Moreover, we identify DNA methyltransferase 3ba (Dnmt3ba) as the primary enzyme responsible for the deposition of this mCH mark. Finally, we observe that TGCT-methylated repeats are specifically associated with H3K9me3-marked heterochromatin suggestive of a functional interplay between these two gene-regulatory marks. Altogether, this work provides insight into a novel form of vertebrate mCH and highlights the substrate diversity of vertebrate DNA methyltransferases.

Functional Diversification of SRSF Protein Kinase to Control Ubiquitin-Dependent Neurodevelopmental Signaling.

SummaryConserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.

Functional Diversification of Ser-Arg Rich Protein Kinases to Control Ubiquitin-Dependent Neurodevelopmental Signalling

Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. Ser-Arg Repeat Protein Kinase (SRPK) is one such conserved eukaryotic kinase, which controls mRNA splicing. Surprisingly, we show that SRPK has acquired a novel function in regulating a neurodevelopmental ubiquitin signalling pathway. In mammalian embryonic stem cells, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of transcription factor substrates, thereby acting to restrain a neural gene expression programme that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signalling that ensures correct regulation of neurodevelopmental gene expression.

Generation and applications of mammalian haploid embryonic stem cells

Mammalian embryonic stem cells (ESCs) are usually diploid, which greatly limit their applications in genetic studies due to existence of two copies of each gene. Recently, the haploid embryonic stem cells (haESCs) have been established from different species, enabling large-scale forward or reverse genetic screening in mammals. The haESCs, same as diploid ESCs, sustain the abilities of self-renewal, proliferation and differentiation, greatly enhancing genetic analyses at the cellular level. Meanwhile, the allodiploid ESCs derived by fusion of haESCs from mice and rats provide a unique system to study the evolution of genes. Besides, the ability of mouse haESCs that can be used as sperm replacement for generation of animals opens new avenues for functional genetics in vivo , such as investigating the roles of imprinted genes during prenatal embryonic development, generation of mouse models carrying complex gene modifications to mimic human diseases and screening for critical genes or amino acids of a specific protein involved in a developmental process of interest. Genome Tagging Project (GTP) proposed based on haESCs was initiated recently with the goal to tag every protein in haESCs, followed by generation of tagged mice at genome-wide scale. GTP will provide a standard platform for the protein study, which may enable precise description of protein atlas and regulatory networks during embryonic development.

Bivalency in Drosophila embryos is associated with strong inducibility of Polycomb target genes

ABSTRACTPolycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation is bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. Here, we provide evidence for the existence of bivalency in fly embryos. Using a recently described PcG reporter fly line, we observed a strong reporter inducibility in the embryo and its sharp decrease in larval and adult stages. Analysis of the chromatin landscape of the reporter revealed a strong signal for the repressive PcG mark, H3K27me3, in all three developmental stages and, surprisingly, a strong signal for a transcriptionally activating H3K4me3 mark in the embryo. Using re-chromatin immunoprecipitation experiments, bivalent domains were also uncovered at endogenous PcG targets like the Hox genes.

PACT increases mammalian embryonic neural stem cell properties by facilitating activation of the notch signaling pathway

The protein activator of protein kinase R (PKR) (PACT) is known to play important roles in PKR regulation and microRNA biogenesis. Based on the observation that PACT is specifically expressed in the ventricular zone (VZ) at the mid-neurogenic period, we examine the role of PACT in this embryonic neural stem cell niche. Here, we provide the first evidence that PACT increases neurosphere formation, as well as expression of Notch target genes and the neural stem cell marker Sox2 in primary neural stem cells in vitro. Consistently, introduction of PACT into the mouse embryonic brain in utero increased the fraction of cells localizing to the VZ. We also show that the PACT-enhanced stemness of neural stem cells is PKR-independent. At the molecular level, PACT was revealed to physically interact with C promoter binding factor 1 (CBF1) and dramatically strengthen the association between CBF1 and Notch intracellular domain (NICD), which indicates stabilization of the Notch transcriptional coactivation complex responsible for Notch target gene expression. Taken together, our study indicates that PACT is a novel transcriptional coactivator of the Notch pathway playing a pivotal role during mammalian brain development.

Chicken Embryonic-Stem Cells Are Permissive to Poxvirus Recombinant Vaccine Vectors.

The discovery of mammalian pluripotent embryonic stem cells (ESC) has revolutionised cell research and regenerative medicine. More recently discovered chicken ESC (cESC), though less intensively studied, are increasingly popular as vaccine substrates due to a dearth of avian cell lines. Information on the comparative performance of cESC with common vaccine viruses is limited. Using RNA-sequencing, we compared cESC transcriptional programmes elicited by stimulation with chicken type I interferon or infection with vaccine viruses routinely propagated in primary chicken embryo fibroblasts (CEF). We used poxviruses (fowlpox virus (FWPV) FP9, canarypox virus (CNPV), and modified vaccinia virus Ankara (MVA)) and a birnavirus (infectious bursal disease virus (IBDV) PBG98). Interferon-stimulated genes (ISGs) were induced in cESC to levels comparable to those in CEF and immortalised chicken fibroblast DF-1 cells. cESC are permissive (with distinct host transcriptional responses) to MVA, FP9, and CNPV but, surprisingly, not to PBG98. MVA, CNPV, and FP9 suppressed innate immune responses, while PBG98 induced a subset of ISGs. Dysregulation of signalling pathways (i.e., NFκB, TRAF) was observed, which might affect immune responses and viral replication. In conclusion, we show that cESC are an attractive alternative substrate to study and propagate poxvirus recombinant vaccine vectors.

124 Functional analysis of porcine OCT4 transcriptional regulatory region-based reporter system

Gene OCT4 plays pivotal roles in maintaining pluripotency of early mammalian embryonic development and embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region for the study of pluripotency. However, there is still a lack of sufficient information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, first, we conducted an investigation to find conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. A similarity of nucleotide sequences of the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and 4 regulatory regions including distal and proximal enhancer elements have a high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay indicated that the porcine OCT4 distal enhancer and proximal enhancer are highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively (n=3). Comparison analysis of naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, Klf2) or primed (Gata6, Mixl1, Fgf5, Otx2) state marker gene expression in a dual-reporter assay using pOCT4-DE-eGFP and pOCT4-PE-DsRed2 showed that expression of naïve and primed markers were up-regulated in cells with high green fluorescent protein and red fluorescent protein expression, respectively (n=3). Porcine OCT4-upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work could provide basic information for the porcine OCT4 upstream region and the various porcine OCT4-fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and for the establishment of embryonic stem cells in pigs. This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Development of High Value-Added Food Technology Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 118042-03-1-HD020).