Establishment Of Pluripotency Research Articles

Maternal Nanog is required for zebrafish embryo architecture and for cell viability during gastrulation.

Nanog has been implicated in establishment of pluripotency in mammals and in zygotic genome activation in zebrafish. In this study, we characterize the development of MZnanog (maternal and zygotic null) mutant zebrafish embryos. Without functional Nanog, epiboly is severely affected, embryo axes do not form and massive cell death starts at the end of gastrulation. We show that three independent defects in MZnanog mutants contribute to epiboly failure: yolk microtubule organization required for epiboly is abnormal, maternal mRNA fails to degrade owing to the absence of miR-430, and actin structure of the yolk syncytial layer does not form properly. We further demonstrate that the cell death in MZnanog embryos is cell-autonomous. Nanog is necessary for correct spatial expression of the ventral-specifying genes bmp2b, vox and vent, and the neural transcription factor her3 It is also required for the correctly timed activation of endoderm genes and for the degradation of maternal eomesa mRNA via miR-430. Our findings suggest that maternal Nanog coordinates several gene regulatory networks that shape the embryo during gastrulation.

Kdm2b Regulates Somatic Reprogramming through Variant PRC1 Complex-Dependent Function

Polycomb repressive complex 1 (PRC1) plays essential roles in cell-fate determination. Recent studies have found that the composition of mammalian PRC1 is particularly varied and complex; however, little is known about the functional consequences of these variant PRC1 complexes on cell-fate determination. Here, we show that Kdm2b promotes Oct4-induced somatic reprogramming through recruitment of a variant PRC1 complex (PRC1.1) to CpG islands (CGIs). Furthermore, we find that bone morphogenetic protein (BMP) represses Oct4/Kdm2b-induced somatic reprogramming selectively. Mechanistically, BMP-SMAD pathway attenuates PRC1.1 occupation and H2AK119 ubiquitination at genes linked to development, resulting in the expression of mesendodermal factors such as Sox17 and a consequent suppression of somatic reprogramming. These observations reveal that PRC1.1 participates in the establishment of pluripotency and identify BMP4 signaling as a modulator of PRC1.1 function.

An interview with Jenny Nichols.

Jennifer Nichols is a Principal Investigator at the Cambridge Stem Cell Institute and Department of Physiology, Development and Neuroscience at the University of Cambridge, UK. Her lab works on lineage segregation and the establishment of pluripotency in the mammalian embryo. In 2017 she was awarded the British Society for Developmental Biology's Cheryll Tickle Medal, given to mid-career female scientists with outstanding achievements in developmental biology. We met Jenny in her Cambridge office to talk about pluripotency in vitro and in vivo, the importance of collaboration in her career path, and what playing a musical instrument has in common with research.

NAC1 Regulates Somatic Cell Reprogramming by Controlling Zeb1 and E-cadherin Expression.

SummaryReprogramming somatic cells to induced pluripotent stem cells (iPSCs) is a long and inefficient process. A thorough understanding of the molecular mechanisms underlying reprogramming is paramount for efficient generation and safe application of iPSCs in medicine. While intensive efforts have been devoted to identifying reprogramming facilitators and barriers, a full repertoire of such factors, as well as their mechanistic actions, is poorly defined. Here, we report that NAC1, a pluripotency-associated factor and NANOG partner, is required for establishment of pluripotency during reprogramming. Mechanistically, NAC1 is essential for proper expression of E-cadherin by a dual regulatory mechanism: it facilitates NANOG binding to the E-cadherin promoter and fine-tunes its expression; most importantly, it downregulates the E-cadherin repressor ZEB1 directly via transcriptional repression and indirectly via post-transcriptional activation of the miR-200 miRNAs. Our study thus uncovers a previously unappreciated role for the pluripotency regulator NAC1 in promoting efficient somatic cell reprogramming.

DEAD-Box RNA Binding Protein DDX5: Not a Black-Box during Reprogramming

The role of RNA binding proteins (RBPs) during nuclear reprogramming is poorly characterized. In this issue of Cell Stem Cell,Li etal. (2017) show that DEAD-box RBP DDX5 acts as a reprogramming roadblock and give important mechanistic insights into the establishment of pluripotency by characterizing the intricate downstream events.

The SIN3A/HDAC Corepressor Complex Functionally Cooperates with NANOG to Promote Pluripotency

Although SIN3A is required for the survival of early embryos and embryonic stem cells (ESCs), the role of SIN3A in the maintenance and establishment of pluripotency remains unclear. Here, we find that theSIN3A/HDAC corepressor complex maintains ESC pluripotency and promotes the generation of induced pluripotent stem cells (iPSCs). Members of the SIN3A/HDAC corepressor complex are enriched in an extended NANOG interactome and function in transcriptional coactivation in ESCs. We also identified a critical role for SIN3A and HDAC2 in efficient reprogramming of somatic cells. Mechanistically, NANOG and SIN3A co-occupy transcriptionally active pluripotency genes in ESCs and also co-localize extensively at their genome-wide targets in pre-iPSCs. Additionally, both factors are required to directly induce a synergistic transcriptional program wherein pluripotency genes are activated and reprogramming barrier genes are repressed. Our findings indicate a transcriptional regulatory role for amajor HDAC-containing complex in promoting pluripotency.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming.

ABSTRACTThe regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.

Disruption of the UBF gene induces aberrant somatic nucleolar bodies and disrupts embryo nucleolar precursor bodies

The nucleolus is the site of ribosome biogenesis and forms around the actively transcribed ribosomal RNA (rRNA) genes. However, the nucleolus is also implicated in cell cycle regulation, tumour suppression and chromosome segregation and nucleolar disfunction is linked to a wide range of human diseases. Interestingly, the nucleolus is also required for genome reprogramming and the establishment of heterochromatin in the mammalian embryo. Mammalian oocytes contain a subnuclear structure that is believed to be the precursor of the functional nucleolus, the Nucleolar Precursor Body (NPB). But the NPB is also required for the organisation of the zygotic heterochromatin and the establishment of pluripotency. We found that disruption of the mouse Upstream Binding Factor (UBF (UBTF)) gene caused disassembly of somatic nucleoli and the accumulation of the key rRNA gene transcription factors into dense subnuclear foci resembling NPBs. Here we show that UBF deletion causes the rRNA genes to collapse onto their centromere-proximal chromosomal sites spatially distinct from NPB-like structures, and that these structures contain rRNA gene transcription factors but not all nucleolar proteins. We further find that embryonic NPBs and their surrounding heterochromatin are both disrupted in UBF-null mouse embryos. These embryos also display subnuclear foci containing the rRNA gene transcription factors and arrest development before completing the forth cleavage division. The data suggest that the rRNA gene transcription factors have an intrinsic ability to interact and form a discrete nuclear compartment even in the absence of any rRNA gene activity and that the formation or maintenance of the zygotic NPB and surrounding heterochromatin requires UBF.

Metabolic restructuring and cell fate conversion.

Accumulating evidence implicates mitochondrial and metabolic pathways in the establishment of pluripotency, as well as in the control of proliferation and differentiation programs. From classic studies in mouse embryos to the latest findings in adult stem cells, human embryonic and induced pluripotent stem cells, an increasing number of evidence suggests that mitochondrial and metabolic-related processes might intertwine with signaling networks and epigenetic rewiring, thereby modulating cell fate decisions. This review summarizes the progresses in this exciting field of research. Dissecting these complex mitochondrial and metabolic mechanisms may lead to a more comprehensive understanding of stemness biology and to potential improvements in stem cell applications for biomedicine, cell therapy, and disease modeling.

345 LOSS OF Brm AND Baf170, COMPONENTS OF THE ATP-DEPENDENT CHROMATIN REMODELING COMPLEX, FACILITATES REPROGRAMMING OF SOMATIC CELL

The SWI/SNF [SWItch/Sucrose NonFermentable; also known as BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodelling complexes are epigenetic modifiers that change the structure of chromatin, and thereby modulate gene transcription. The BAF chromatin remodeling complexes undergo progressive changes in subunit composition during cellular differentiation. In embryonic stem cells (ESC), BAF complex, esBAF, contains Brg1 and Baf155, which are crucial for ESC self-renewal and also facilitate induced pluripotent stem cell (iPSC) reprogramming from somatic cells. Here we sought to determine the roles of somatic BAF components (Brm and Baf170, homologues of Brg1 and Baf155, respectively) in mouse iPSC reprogramming through shRNA-mediated knockdown studies. We found that the expression of Brm and Baf170 were inhibited in reprogramming by Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) activity, which is essential for pluripotency establishment. We further found that knockdown of Brm and Baf170 in mouse somatic cells promotes reprogramming efficiency. Specifically, loss of Brm and Baf170 during early (Days 3 and 6 after initial iPSC induction) and later-stage (Day 9) reprogramming, respectively, improves the numbers of iPSC colonies formed. These also led to significant upregulation of pluripotent genes, including Sox2, Nanog, Fgf5, and Tbx3. Although the somatic Brm and Baf170 are believed to be absent in ESC, the expression levels of Nanog and Tbx3 were increased significantly by knockdown of either Brm or Baf170 in ESC. Finally, we showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed cells (pre-iPSC). These data suggest that inhibiting somatic BAF complex improved complete reprogramming by facilitating the activation of the pluripotency circuitry. A reduced activity of the somatic BAF complex constitutes part of the Stat3 regulated epigenetic changes during pluripotency establishment.

Pelota regulates the development of extraembryonic endoderm through activation of bone morphogenetic protein (BMP) signaling

Pelota (Pelo) is ubiquitously expressed, and its genetic deletion in mice leads to embryonic lethality at an early post-implantation stage. In the present study, we conditionally deleted Pelo and showed that PELO deficiency did not markedly affect the self-renewal of embryonic stem cells (ESCs) or their capacity to differentiate in teratoma assays. However, their differentiation into extraembryonic endoderm (ExEn) in embryoid bodies (EBs) was severely compromised. Conversely, forced expression of Pelo in ESCs resulted in spontaneous differentiation toward the ExEn lineage. Failure of Pelo-deficient ESCs to differentiate into ExEn was accompanied by the retained expression of pluripotency-related genes and alterations in expression of components of the bone morphogenetic protein (BMP) signaling pathway. Further experiments have also revealed that attenuated activity of BMP signaling is responsible for the impaired development of ExEn. The recovery of ExEn and down-regulation of pluripotent genes in BMP4-treated Pelo-null EBs indicate that the failure of mutant cells to down-regulate pluripotency-related genes in EBs is not a result of autonomous defect, but rather to failed signals from surrounding ExEn lineage that induce the differentiation program. In vivo studies showed the presence of ExEn in Pelo-null embryos at E6.5, yet embryonic lethality at E7.5, suggesting that PELO is not required for the induction of ExEn development, but rather for ExEn maintenance or for terminal differentiation toward functional visceral endoderm which provides the embryos with growth factors required for further development. Moreover, Pelo-null fibroblasts failed to reprogram toward induced pluripotent stem cells (iPSCs) due to inactivation of BMP signaling and impaired mesenchymal-to-epithelial transition. Thus, our results indicate that PELO plays an important role in the establishment of pluripotency and differentiation of ESCs into ExEn lineage through activation of BMP signaling.

Pou5f1/Oct4 Promotes Cell Survival via Direct Activation of mych Expression during Zebrafish Gastrulation

Myc proteins control cell proliferation, cell cycle progression, and apoptosis, and play important roles in cancer as well in establishment of pluripotency. Here we investigated the control of myc gene expression by the Pou5f1/Oct4 pluripotency factor in the early zebrafish embryo. We analyzed the expression of all known zebrafish Myc family members, myca, mycb, mych, mycl1a, mycl1b, and mycn, by whole mount in situ hybridization during blastula and gastrula stages in wildtype and maternal plus zygotic pou5f1 mutant (MZspg) embryos, as well as by quantitative PCR and in time series microarray data. We found that the broad blastula and gastrula stage mych expression, as well as late gastrula stage mycl1b expression, both depend on Pou5f1 activity. We analyzed ChIP-Seq data and found that both Pou5f1 and Sox2 bind to mych and mycl1b control regions. The regulation of mych by Pou5f1 appears to be direct transcriptional activation, as overexpression of a Pou5f1 activator fusion protein in MZspg embryos induced strong mych expression even when translation of zygotically expressed mRNAs was suppressed. We further showed that MZspg embryos develop enhanced apoptosis already during early gastrula stages, when apoptosis was not be detected in wildtype embryos. However, Mych knockdown alone did not induce early apoptosis, suggesting potentially redundant action of several early expressed myc genes, or combination of several pathways affected in MZspg. Experimental mych overexpression in MZspg embryos did significantly, but not completely suppress the apoptosis phenotype. Similarly, p53 knockdown only partially suppressed apoptosis in MZspg gastrula embryos. However, combined knockdown of p53 and overexpression of Mych completely rescued the MZspg apoptosis phenotype. These results reveal that Mych has anti-apoptotic activity in the early zebrafish embryo, and that p53-dependent and Myc pathways are likely to act in parallel to control apoptosis at these stages.

Nanog-Independent Reprogramming to iPSCs with Canonical Factors

SummaryIt has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem cells and induced pluripotent stem cells (iPSCs). However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever-increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical-reprogramming factors Oct4, Sox2, Klf4, and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog−/− fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPSCs from Nanog−/− fibroblasts that effectively contributed to the germline of chimeric mice. Thus, whereas Nanog may be an important mediator of reprogramming, it is not required for establishing pluripotency in the mouse, even under standard conditions.

Do all roads lead to Oct4? The emerging concepts of induced pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Roles for MYC in the Establishment and Maintenance of Pluripotency

MYC and MYCN have been directly implicated in the transcriptional regulation of several thousand genes in pluripotent stem cells and possibly contribute to the activity of all transcribed genes. Control of transcription by a pause-release mechanism, recruitment of positive and negative epigenetic regulators, and a general role in transcriptional amplification have all been implicated as part of the broad, overarching mechanism by which MYC controls stem cell biology. As would be anticipated from the regulation of so many genes, MYC is involved in a wide range of cellular processes including cell-cycle control, metabolism, signal transduction, self-renewal, maintenance of pluripotency, and control of cell fate decisions. MYC transcription factors also have clear roles in cell reprogramming and establishment of the pluripotent state. The mechanism by which MYC accomplishes this is now being explored and promises to uncover unexpected facets of general MYC regulation that are likely to be applicable to cancer biology. In this work we review our current understanding of how MYC contributes to the maintenance and establishment of pluripotent cells and how it contributes to early embryonic development.

The THO Complex Regulates Pluripotency Gene mRNA Export and Controls Embryonic Stem Cell Self-Renewal and Somatic Cell Reprogramming

Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, posttranscriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5 and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, because its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of posttranscriptional regulation in stem cell fate specification.

Oct4 Cell-Autonomously Promotes Primitive Endoderm Development in the Mouse Blastocyst

In embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.

Network Theory Inspired Analysis of Time-Resolved Expression Data Reveals Key Players Guiding P. patens Stem Cell Development

Transcription factors (TFs) often trigger developmental decisions, yet, their transcripts are often only moderately regulated and thus not easily detected by conventional statistics on expression data. Here we present a method that allows to determine such genes based on trajectory analysis of time-resolved transcriptome data. As a proof of principle, we have analysed apical stem cells of filamentous moss (P. patens) protonemata that develop from leaflets upon their detachment from the plant. By our novel correlation analysis of the post detachment transcriptome kinetics we predict five out of 1,058 TFs to be involved in the signaling leading to the establishment of pluripotency. Among the predicted regulators is the basic helix loop helix TF PpRSL1, which we show to be involved in the establishment of apical stem cells in P. patens. Our methodology is expected to aid analysis of key players of developmental decisions in complex plant and animal systems.

NANOG-dependent function of TET1 and TET2 in establishment of pluripotency.

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein Nanog1–2, which plays an essential role in establishing ground state pluripotency during somatic cell reprogramming3–4. While the genomic occupancy of Nanog has been extensively investigated, comparatively little is known about Nanog-associated proteins5 and their contribution to the Nanog-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identified 27 high-confidence protein interaction partners of Nanog in mouse ES cells. These consist of 19 novel partners of Nanog that have not been reported before including the Ten eleven translocation (Tet) family methylcytosine hydroxylase Tet1. We confirmed physical association of Nanog with Tet1, and demonstrated that Tet1, in synergy with Nanog, enhances the efficiency of reprogramming. We also found physical association and reprogramming synergy of Tet2 with Nanog, and demonstrated that knockdown of Tet2 abolishes the reprogramming synergy of Nanog with a catalytically deficient mutant of Tet1 (Tet1Mut). These results indicate that the physical interaction between Nanog and Tet1/2 proteins facilitates reprogramming in a manner that is dependent on Tet1/2's catalytic activity. Tet1 and Nanog co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in ES cells, and Tet1 binding is reduced upon Nanog depletion. Co-expression of Nanog and Tet1 results in expression priming of and increased 5hmC levels at top ranked common targets Esrrb and Oct4 before reprogramming to naïve pluripotency. We propose that Tet1 is recruited by Nanog to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of Nanog and uncover a novel role for 5mC hydroxylases in the establishment of naïve pluripotency.

278 Jak/Stat3 SIGNALING PROMOTES SOMATIC CELL REPROGRAMMING BY EPIGENETIC REGULATION

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSC) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remains unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells, and the partially reprogrammed cells (pre-iPSC). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEF). We demonstrated that activated Stat3 not only promotes but is essential for the pluripotency establishment in MEF cell reprogramming. We further demonstrated that, during reprogramming, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory DNA sequences in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDAC) expression, as well as the increased expression of lysine-specific histone demethylase 2 and methyl-CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSC. We conclude that Jak/Stat3 activity plays a fundamental role in promoting the establishment of pluripotency at the epigenetic level, by facilitating DNA demethylation/de novo methylation and open-chromatin formation during late stage reprogramming.