Pluripotency Of Mouse Embryonic Stem Cells Research Articles

LIF maintains mouse embryonic stem cells pluripotency by modulating TET1 and JMJD2 activity in a JAK2-dependent manner.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.

BACH1 recruits NANOG and histone H3 lysine 4 methyltransferase MLL/SET1 complexes to regulate enhancer-promoter activity and maintains pluripotency.

Maintenance of stem-cell identity requires proper regulation of enhancer activity. Both transcription factors OCT4/SOX2/NANOG and histone methyltransferase complexes MLL/SET1 were shown to regulate enhancer activity, but how they are regulated in embryonic stem cells (ESCs) remains further studies. Here, we report a transcription factor BACH1, which directly interacts with OCT4/SOX2/NANOG (OSN) and MLL/SET1 methyltransferase complexes and maintains pluripotency in mouse ESCs (mESCs). BTB domain and bZIP domain of BACH1 are required for these interactions and pluripotency maintenance. Loss of BACH1 reduced the interaction between NANOG and MLL1/SET1 complexes, and decreased their occupancy on chromatin, and further decreased H3 lysine 4 trimethylation (H3K4me3) level on gene promoters and (super-) enhancers, leading to decreased enhancer activity and transcription activity, especially on stemness-related genes. Moreover, BACH1 recruited NANOG through chromatin looping and regulated remote NANOG binding, fine-tuning enhancer–promoter activity and gene expression. Collectively, these observations suggest that BACH1 maintains pluripotency in ESCs by recruiting NANOG and MLL/SET1 complexes to chromatin and maintaining the trimethylated state of H3K4 and enhancer–promoter activity, especially on stemness-related genes.

Targeting the histone demethylase LSD1 prevents cardiomyopathy in a mouse model of laminopathy.

LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.

Long noncoding RNA lnc-NAP sponges mmu-miR-139-5p to modulate Nanog functions in mouse ESCs and embryos

ABSTRACT The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3′ untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.

Babam2 Regulates Cell Cycle Progression and Pluripotency in Mouse Embryonic Stem Cells as Revealed by Induced DNA Damage.

BRISC and BRCA1-A complex member 2 (Babam2) plays an essential role in promoting cell cycle progression and preventing cellular senescence. Babam2-deficient fibroblasts show proliferation defect and premature senescence compared with their wild-type (WT) counterpart. Pluripotent mouse embryonic stem cells (mESCs) are known to have unlimited cell proliferation and self-renewal capability without entering cellular senescence. Therefore, studying the role of Babam2 in ESCs would enable us to understand the mechanism of Babam2 in cellular aging, cell cycle regulation, and pluripotency in ESCs. For this study, we generated Babam2 knockout (Babam2−/−) mESCs to investigate the function of Babam2 in mESCs. We demonstrated that the loss of Babam2 in mESCs leads to abnormal G1 phase retention in response to DNA damage induced by gamma irradiation or doxorubicin treatments. Key cell cycle regulators, CDC25A and CDK2, were found to be degraded in Babam2−/− mESCs following gamma irradiation. In addition, Babam2−/− mESCs expressed p53 strongly and significantly longer than in control mESCs, where p53 inhibited Nanog expression and G1/S cell cycle progression. The combined effects significantly reduced developmental pluripotency in Babam2−/− mESCs. In summary, Babam2 maintains cell cycle regulation and pluripotency in mESCs in response to induced DNA damage.

Abscission Couples Cell Division to Embryonic Stem Cell Fate.

SummaryCell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cell types, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here, we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs naive pluripotency exit, and artificially inducing abscission accelerates it. Altogether, our data indicate that a switch in the division machinery leading to faster abscission regulates pluripotency exit. Our study identifies abscission as a key cellular process coupling cell division to fate transitions.

β-Catenin and Associated Proteins Regulate Lineage Differentiation in Ground State Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. β-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of β-catenin in restraining germline and somatic lineage differentiation genes. We show that β-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help β-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of β-catenin in preserving lineage differentiation integrity in ground state ESCs.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states invitro.

The Long Non-coding RNA Cyrano Is Dispensable for Pluripotency of Murine and Human Pluripotent Stem Cells.

SummaryPluripotency is tightly regulated and is crucial for stem cells and their implementation for regenerative medicine. Non-coding RNAs, especially long non-coding RNAs (lncRNAs) emerged as orchestrators of versatile (patho)-physiological processes on the transcriptional and post-transcriptional level. Cyrano, a well-conserved lncRNA, is highly expressed in stem cells suggesting an important role in pluripotency, which we aimed to investigate in loss-off-function (LOF) experiments. Cyrano was described previously to be essential for the maintenance of mouse embryonic stem cell (ESC) pluripotency. In contrast, using different genetic models, we here found Cyrano to be dispensable in murine and human iPSCs and in human ESCs. RNA sequencing revealed only a moderate influence of Cyrano on the global transcriptome. In line, Cyrano-depleted iPSCs retained the potential to differentiate into the three germ layers. In conclusion, different methods were applied for LOF studies to rule out potential off-target effects. These approaches revealed that Cyrano does not impact pluripotency.

Prediction model construction of mouse stem cell pluripotency using CpG and non-CpG DNA methylation markers

BackgroundGenome-wide studies of DNA methylation across the epigenetic landscape provide insights into the heterogeneity of pluripotent embryonic stem cells (ESCs). Differentiating into embryonic somatic and germ cells, ESCs exhibit varying degrees of pluripotency, and epigenetic changes occurring in this process have emerged as important factors explaining stem cell pluripotency.ResultsHere, using paired scBS-seq and scRNA-seq data of mice, we constructed a machine learning model that predicts degrees of pluripotency for mouse ESCs. Since the biological activities of non-CpG markers have yet to be clarified, we tested the predictive power of CpG and non-CpG markers, as well as a combination thereof, in the model. Through rigorous performance evaluation with both internal and external validation, we discovered that a model using both CpG and non-CpG markers predicted the pluripotency of ESCs with the highest prediction performance (0.956 AUC, external test). The prediction model consisted of 16 CpG and 33 non-CpG markers. The CpG and most of the non-CpG markers targeted depletions of methylation and were indicative of cell pluripotency, whereas only a few non-CpG markers reflected accumulations of methylation. Additionally, we confirmed that there exists the differing pluripotency between individual developmental stages, such as E3.5 and E6.5, as well as between induced mouse pluripotent stem cell (iPSC) and somatic cell.ConclusionsIn this study, we investigated CpG and non-CpG methylation in relation to mouse stem cell pluripotency and developed a model thereon that successfully predicts the pluripotency of mouse ESCs.

The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells.

Mammals optimize their physiology to the light-dark cycle by synchronization of the master circadian clock in the brain with peripheral clocks in the rest of the tissues of the body. Circadian oscillations rely on a negative feedback loop exerted by the molecular clock that is composed by transcriptional activators Bmal1 and Clock, and their negative regulators Period and Cryptochrome. Components of the molecular clock are expressed during early development, but onset of robust circadian oscillations is only detected later during embryogenesis. Here, we have used naïve pluripotent mouse embryonic stem cells (mESCs) to study the role of Bmal1 during early development. We found that, compared to wild-type cells, Bmal1-/- mESCs express higher levels of Nanog protein and altered expression of pluripotency-associated signalling pathways. Importantly, Bmal1-/- mESCs display deficient multi-lineage cell differentiation capacity during the formation of teratomas and gastrula-like organoids. Overall, we reveal that Bmal1 regulates pluripotent cell differentiation and propose that the molecular clock is an hitherto unrecognized regulator of mammalian development.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice.

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.

Dicer as a Repressor of Antiviral Responses in Mouse Embryonic Stem Cells

Recent studies have demonstrated that pluripotent stem cells (PSCs), including human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have underdeveloped antiviral innate immunity. Specifically, they fail to express IFN in response to viral stimuli. This finding has led to the hypothesis that the lack of IFN response is an intrinsic property of PSCs. It has also inspired the investigation of the underlying molecular mechanisms. In an effort to conduct a holistic analysis of antiviral responses in ESCs, we have developed an assay that can simultaneously measure RNA‐mediated gene expression and RNA‐induced antiviral responses. In this system, GFP is expressed from functionalized synthetic GFP‐mRNA and is used to measure the cellular translation activity, whereas dsRNA and 5’‐triphosphate‐ssRNA act as viral RNA analogs. Transfection of ESCs with these synthetic RNAs can potently activate all common antiviral signaling pathways, including RNA interference (RNAi), dsRNA activated protein kinase (PKR), OAS‐RNase L, and IFN responses. Our data revealed that RNAi activity was detected in ESCs, but not in Dicer knockout ESCs (D‐/‐ESCs) as expected since Dicer is a key enzyme in miRNA and siRNA processing. The antiviral responses mediated by PKR, OAS‐RNase L, and IFN were either not detected or detected at low levels in ESCs, but they increased dramatically in D‐/‐ESCs, as indicated by increased activity of PKR (measured by low level GFP expression and PKR phosphorylation) and RNase L (determined by RNA degradation). Furthermore, the components of the IFN signaling pathway are expressed at higher basal levels in D‐/‐ESCs than in normal ESCs and are further up‐regulated by RNA transfection. The most notable effect of RNA transfection into D‐/‐ESCs is the induction of cell death and decreased cell proliferation, which is at least partly attributed to PKR activation and increased expression of the cell cycle inhibitors p21 and p19. Our findings suggest that ESCs are exquisitely sensitive to cellular damages caused by antiviral response and that Dicer acts as a repressor of antiviral responses in ESCs under normal conditions.Support or Funding InformationThis work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).

Derivation of LIF-Independent Embryonic Stem Cells Using Inducible OCT4 Expression.

Pluripotent mouse embryonic stem (ES) cells, which are derived from the inner cell mass (ICM) of preimplantation stage embryos, are capable of self-renewing indefinitely in the presence of the external signal leukemia inhibitory factor (LIF), activation of Wnt signaling through inhibition of GSK3, and inhibition of MAP kinase/ERK kinase signaling. The OCT4 transcription factor is expressed highly in pluripotent cells and is a central transcriptional regulator of the pluripotent state. Here, we describe a protocol to culture ES cells in LIF-independent and serum-free media using an inducible OCT4 (iOCT4) ES cell model system. This protocol is sufficient to sustain ES cell self-renewal in vitro in defined conditions in the absence of external signals. LIF-independent iOCT4 ES cells are fully capable of differentiating following deactivation of the inducible OCT4 transgene.

45 Expression patterns of PRDM family genes in porcine pre-implantation embryos

Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values<0.05 were considered significant. There was a significant decrease in PRDM2 expression in 4-cell and blastocyst, PRDM4 expression in 4-cell, and PRDM6 in all stages (MII, zygote, 4-cell, and blastocyst), compared with the GV stage. Because zygotic genome activation occurs at the 4-cell stage in the pig, the significant decrease in gene expression (PRDM2, PRDM4, and PRDM6) indicates they may be maternally originated and involved in the reprogramming process following fertilisation. On the other hand, there was a significant increase in PRDM15 expression in blastocysts and the PRDM14 transcript was only detected in blastocysts in all three biological replicates, suggesting that the genes are most likely involved in pluripotency maintenance, as was found in previous human studies. These results indicate that PRDM family genes are differentially expressed during early embryo development in pigs and may play a role in maintenance of pluripotency. For further study, we intend to evaluate the role of PRDM family genes during early embryo development in pigs.

The effect of lead during the Flint water crisis on mouse embryonic stem cells self-renewal and differentiation markers

During the Flint water crisis, the residents of Flint, Michigan experienced a significant increase in blood lead levels. For some this resulted in an increase as high as 40 μg/dL from 5 μg/dL, which is considered to be safe by the Center for Disease Control and Prevention. Since the extent of the effect of the lead exposure in early embryonic development is not greatly investigated, the aim of this study is to explore the effect of lead exposure at concentrations present in Flint, MI during the Flint water crisis in the embryonic development. The expression of pluripotency and self-renewal markers (Oct4, Sox2, Nanog and Zfp-42) coupled with morphological and alkaline phosphatase assays revealed that mouse embryonic stem cells (mESC) pluripotency and self-renewal capabilities are perturbed following exposure in a lead acetate concentration dependent manner. Moreover, mouse embryoid bodies (mEB), which provide ideal models for testing toxicity in vitro, revealed that lead acetate exposure induces fewer but larger mEBs, whereas gene expression analysis of lineage specific transcription factors showed an increased mRNA level of endodermal (Gata 4, Gata 6, Sox 7) and mesodermal markers (Eomes, Hand 1, Slug 1) while the mRNA level of ectodermal markers (Otx 2, Noggin, Sox 1) decreased. Taken all together, these results indicate that lead acetate disturbs the pluripotency of mESC and differentiation potential of mEBs by inhibiting differentiation towards ectodermal lineages and inducing it towards endodermal and mesodermal lineages.

The long non-coding RNA Snhg3 is essential for mouse embryonic stem cell self-renewal and pluripotency

BackgroundSmall nucleolar RNA host gene 3 (Snhg3) is a long non-coding RNA (lncRNA) that was shown to participate in the tumorigenesis of certain cancers. However, little is known about its role in embryonic stem cells (ESCs).MethodsHere, we investigated the role of Snhg3 in mouse ESCs (mESCs) through both loss-of-function (knockdown) and gain-of-function (overexpression) approaches. Alkaline phosphatase staining, secondary colony formation, propidium iodide staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to access self-renewal capacity, whereas immunofluorescence, qRT-PCR, and embryoid body formation were performed to examine pluripotency. In addition, the effect of Snhg3 on mouse embryonic development was determined based on the morphological changes, blastocyst rate, and altered pluripotency marker (Nanog, Oct4) expression. Moreover, the relationship between Snhg3 and key pluripotency factors was evaluated by chromatin immunoprecipitation qPCR, qRT-PCR, subcellular fractionation, and RNA immunoprecipitation. Finally, RNA pull-down and mass spectrometry were applied to explore the potential interacting proteins of Snhg3 in mESCs.ResultsWe demonstrated that Snhg3 is essential for self-renewal and pluripotency maintenance in mESCs. In addition, Snhg3 knockdown disrupted mouse early embryo development. Mechanistically, Snhg3 formed a positive feedback network with Nanog and Oct4, and 126 Snhg3-interacting proteins were identified in mESCs.ConclusionsSnhg3 is essential for mESC self-renewal and pluripotency, as well as mouse early embryo development.

Structurally Tunable Reduced Graphene Oxide Substrate Maintains Mouse Embryonic Stem Cell Pluripotency.

Culturing embryonic stem cells (ESCs) in vitro usually requires animal‐derived trophoblast cells, which may cause pathogenic and immune reactions; moreover, the poor repeatability between batches hinders the clinical application of ESCs. Therefore, it is essential to synthesize a xenogeneic‐free and chemically well‐defined biomaterial substrate for maintaining ESC pluripotency. Herein, the effects of structurally tunable reduced graphene oxide (RGO) substrates with different physicochemical properties on ESC pluripotency are studied. Colony formation and CCK‐8 assays show that the RGO substrate with an average 30 µm pore size promotes cell survival and proliferation. The unannealed RGO substrate promotes ESC proliferation significantly better than the annealed substrate due to the interfacial hydrophilic groups. The RGO substrate can also maintain ESC for a long time. Additionally, immunofluorescence staining shows that ESCs cultured on an RGO substrate highly express E‐cadherin and β‐catenin, whereas after being modified by Dickkopf‐related protein 1, the RGO substrate is unable to sustain ESC pluripotency. Furthermore, the cell line that interferes with E‐cadherin is also unable to maintain pluripotency. These results confirm that the RGO substrate maintains ESC pluripotency by promoting E‐cadherin‐mediated cell–cell interaction and Wnt signaling.

The transcription factor TFCP2L1 induces expression of distinct target genes and promotes self-renewal of mouse and human embryonic stem cells

TFCP2L1 (transcription factor CP2-like 1) is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency. However, the direct TFCP2L1 target genes are uncharacterized. Here, using gene overexpression, immunoblotting, quantitative real-time PCR, ChIP, and reporter gene assays, we show that TFCP2L1 primarily induces estrogen-related receptor β (Esrrb) expression that supports mouse ESC identity and also selectively enhances Kruppel-like factor 4 (Klf4) expression and thereby promotes human ESC self-renewal. Specifically, we found that in mouse ESCs, TFCP2L1 binds directly to the Esrrb gene promoter and regulates its transcription. Esrrb knockdown impaired Tfcp2l1's ability to induce interleukin 6 family cytokine (leukemia inhibitory factor)-independent ESC self-renewal and to reprogram epiblast stem cells to naïve pluripotency. Conversely, Esrrb overexpression blocked differentiation induced by Tfcp2l1 down-regulation. Moreover, we identified Klf4 as a direct TFCP2L1 target in human ESCs, bypassing the requirement for activin A and basic fibroblast growth factor in short-term human ESC self-renewal. Enforced Klf4 expression recapitulated the self-renewal-promoting effect of Tfcp2l1, whereas Klf4 knockdown eliminated these effects and caused loss of colony-forming capability. These findings indicate that TFCP2L1 functions differently in naïve and primed pluripotency, insights that may help elucidate the different states of pluripotency.

Activated MEK/ERK Pathway Drives Widespread and Coordinated Overexpression of UHRF1 and DNMT1 in Cancer cells

The UHRF1-DNMT1 axis plays a key role in DNA maintenance methylation in mammals. Accumulative studies demonstrate that UHRF1 is broadly overexpressed in cancers, which contributes to cancer cell proliferation and tumorigenesis. Interestingly, a proteasome-dependent downregulation of UHRF1 has been observed in pluripotent ground state mouse embryonic stem cells (mESCs) cultured in the presence of two kinase (MEK1/MEK2 and GSK3β) inhibitors (termed 2i), raising the question whether UHRF1 is similarly regulated in cancer cells. Here we present evidence that while addition of 2i broadly downregulates UHRF1 and DNMT1 in various cancer cells, distinct underlying mechanisms are involved. In contrast to mESCs, 2i-induced downregulation of UHRF1 and DNMT1 in cancer cells cannot be rescued by proteasome inhibitor and occurs primarily at the level of transcription. Furthermore, downregulation of UHRF1 and DNMT1 by 2i is due to inhibition of MEK1/MEK2, but not GSK3β activity. Data mining reveals a marked co-expression of UHRF1 and DNMT1 in normal tissues as well as cancers. We provide evidence that multiple transcription factors including E2F1 and SP1 mediate the transcriptional activation of UHRF1 and DNMT1 by the activated MEK/ERK pathway. Together our study reveals distinct regulation of UHRF1/DNMT1 in mESCs and cancer cells and identifies activated MEK/ERK pathway as a driving force for coordinated and aberrant over-expression of UHRF1 and DNMT1 in cancers.