Pluripotency Regulatory Network Research Articles

A multi-omics integrative analysis based on CRISPR screens re-defines the pluripotency regulatory network in ESCs

A comprehensive and precise definition of the pluripotency gene regulatory network (PGRN) is crucial for clarifying the regulatory mechanisms in embryonic stem cells (ESCs). Here, after a CRISPR/Cas9-based functional genomics screen and integrative analysis with other functional genomes, transcriptomes, proteomes and epigenome data, an expanded pluripotency-associated gene set is obtained, and a new PGRN with nine sub-classes is constructed. By integrating the DNA binding, epigenetic modification, chromatin conformation, and RNA expression profiles, the PGRN is resolved to six functionally independent transcriptional modules (CORE, MYC, PAF, PRC, PCGF and TBX). Spatiotemporal transcriptomics reveal activated CORE/MYC/PAF module activity and repressed PRC/PCGF/TBX module activity in both mouse ESCs (mESCs) and pluripotent cells of early embryos. Moreover, this module activity pattern is found to be shared by human ESCs (hESCs) and cancers. Thus, our results provide novel insights into elucidating the molecular basis of ESC pluripotency.

Epiblast-like stem cells established by Wnt/β-catenin signaling manifest distinct features of formative pluripotency and germline competence

Different formative pluripotent stem cells harboring similar functional properties have been recently established to be lineage neutral and germline competent yet have distinct molecular identities. Here, we show that WNT/β-catenin signaling activation sustains transient mouse epiblast-like cells as epiblast-like stem cells (EpiLSCs). EpiLSCs display metastable formative pluripotency with bivalent cellular energy metabolism and unique transcriptomic features and chromatin accessibility. We develop single-cell stage label transfer (scSTALT) to study the formative pluripotency continuum and reveal that EpiLSCs recapitulate a unique developmental period invivo, filling the gap of the formative pluripotency continuum between other published formative stem cells. WNT/β-catenin signaling activation counteracts differentiation effects of activin A and bFGF by preventing complete dissolution of naive pluripotency regulatory network. Moreover, EpiLSCs have direct competence toward germline specification, which is further matured by an FGF receptor inhibitor. Our EpiLSCs can serve as an invitro model for mimicking and studying early post-implantation development and pluripotency transition.

ALKBH5 regulates somatic cell reprogramming in a phase-specific manner.

ABSTRACTEstablishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors [octamer binding transcription factor 4 (OCT4; also known as POU5F1), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4) and cellular myelocytomatosis (c-MYC)] provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA-specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, whereas overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5, mediated through its catalytic activity, in regulating somatic cell reprogramming at the post-transcriptional level. This article has an associated First Person interview with the first author of the paper.

Rbbp4 Suppresses Premature Differentiation of Embryonic Stem Cells.

SummaryPolycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages.

Genetic Signatures of Evolution of the Pluripotency Gene Regulating Network across Mammals.

Mammalian pluripotent stem cells (PSCs) have distinct molecular and biological characteristics among species, but to date we lack a comprehensive understanding of regulatory network evolution in mammals. Here, we carried out a comparative genetic analysis of 134 genes constituting the pluripotency gene regulatory network across 48 mammalian species covering all the major taxonomic groups. We report that mammalian genes in the pluripotency regulatory network show a remarkably high degree of evolutionary stasis, suggesting the conservation of fundamental biological process of mammalian PSCs across species. Nevertheless, despite the overall conservation of the regulatory network, we discovered rapid evolution of the downstream targets of the core regulatory elements and specific amino acid residues that have undergone positive selection. Our data indicate development of lineage-specific pluripotency regulating networks that may explain observed variations in some characteristics of mammalian PSCs. We further revealed that positively selected genes could be associated with species’ unique adaptive characteristics that were not dedicated to regulation of PSCs. These results provide important insight into the evolution of the pluripotency gene regulatory network underlying variations in characteristics of mammalian PSCs.

Long noncoding RNA Q associates with Sox2 and is involved in the maintenance of pluripotency in mouse embryonic stem cells.

Large intergenic noncoding RNAs (lincRNAs) in ESCs may play an important role in the maintenance of pluripotency. The identification of stem cell-specific lincRNAs and their interacting partners will deepen our understanding of the maintenance of stem cell pluripotency. We identified a lincRNA, LincQ, which is specifically expressed in ESCs and is regulated by core pluripotent transcription factors. It was rapidly downregulated during the differentiation process. Knockdown of LincQ in ESCs led to differentiation, downregulation of pluripotency-related genes, and upregulation of differentiation-related genes. We found that exon 1 of LincQ can specifically bind to Sox2. The Soxp region in Sox2, rather than the high mobility group domain, is responsible for LincQ binding. Importantly, the interaction between LincQ and Sox2 is required for the maintenance of pluripotency in ESCs and the transcription of pluripotency genes. Esrrb and Tfcp2l1 are key downstream targets of LincQ and Sox2, since overexpression of Esrrb and Tfcp2l1 can restore the loss of ESC pluripotency that is induced by LincQ depletion. In summary, we found that LincQ specifically interacts with Sox2 and contributes to the maintenance of pluripotency, highlighting the critical role of lincRNA in the pluripotency regulatory network.

Whsc1 links pluripotency exit with mesendoderm specification.

How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.

Alternative splicing links histone modifications to stem cell fate decision

BackgroundUnderstanding the embryonic stem cell (ESC) fate decision between self-renewal and proper differentiation is important for developmental biology and regenerative medicine. Attention has focused on mechanisms involving histone modifications, alternative pre-messenger RNA splicing, and cell-cycle progression. However, their intricate interrelations and joint contributions to ESC fate decision remain unclear.ResultsWe analyze the transcriptomes and epigenomes of human ESC and five types of differentiated cells. We identify thousands of alternatively spliced exons and reveal their development and lineage-dependent characterizations. Several histone modifications show dynamic changes in alternatively spliced exons and three are strongly associated with 52.8% of alternative splicing events upon hESC differentiation. The histone modification-associated alternatively spliced genes predominantly function in G2/M phases and ATM/ATR-mediated DNA damage response pathway for cell differentiation, whereas other alternatively spliced genes are enriched in the G1 phase and pathways for self-renewal. These results imply a potential epigenetic mechanism by which some histone modifications contribute to ESC fate decision through the regulation of alternative splicing in specific pathways and cell-cycle genes. Supported by experimental validations and extended datasets from Roadmap/ENCODE projects, we exemplify this mechanism by a cell-cycle-related transcription factor, PBX1, which regulates the pluripotency regulatory network by binding to NANOG. We suggest that the isoform switch from PBX1a to PBX1b links H3K36me3 to hESC fate determination through the PSIP1/SRSF1 adaptor, which results in the exon skipping of PBX1.ConclusionWe reveal the mechanism by which alternative splicing links histone modifications to stem cell fate decision.

Abstract A91: Stage-specific transcriptome analysis of human pancreatic cancer progression

Abstract Our goals are to discover gene networks that control the progression of pancreatic intraepithelial neoplasias (PanINs) to invasive stage pancreatic ductal adenocarcinoma (PDAC) and to understand how the pluripotency regulatory network can temporarily suppress the transition. Certain cancer cells are amenable to reprogramming to pluripotency by somatic cell nuclear transfer (SCNT) or defined transcription factors (iPS). Prior work showed that SCNT-cancer cells develop into normal appearing blastocysts, but later the cancer genome in SCNT-chimeric mice was reactivated during embryogenesis, re-expressing the cancer phenotype of the lineage from which they originated. We previously showed that when an iPS-like line, designated 10-22, derived from human pancreatic ductal adenocarcinoma (PDAC), was injected into immunodeficient mice, they consistently recapitulated pre-invasive, PanIN to invasive stages of PDAC (Kim et al., 2013 Cell Reports). Specifically, the PDAC-iPS cells differentiated into a ductal, PanIN2/3 epithelium with a collagen basement membrane, followed by progression to an invasive stage phenotype with basement membrane breakdown. Furthermore, the ductal cell lesions were surrounded by stroma that co-differentiated from the pluripotent 10-22 cells, indicative of signaling interactions with the epithelium. With this system, we now ask: 1) What gene regulatory networks for pluripotency can transiently attenuate the cancer genome; 2) Upon loss of pluripotency and differentiation, what are the regulatory network transitions during human PDAC progression? 3) What are the reciprocal signals between the PDAC epithelial cells and their surrounding stromal cells during pancreatic cancer progression? To address these questions, we have genetically tagged the PDAC-iPS 10-22 line with a lentiviral vector expressing a ubiquitous promoter driving GFP and a ductal K19 promoter driving infrared protein (iRFP), so that cells can be retrieved by FACS. While GFP+iRFP- cells include stroma derived from the 10-22 cells, GFP+iRFP+ allows isolation of precursor and invasive stages of PDAC. The tagged lines were injected into immunodeficient mice, the lesions were monitored in vivo by iRFP flourescence, and RNA was isolated in triplicate at multiple time points and subjected to RNA-Seq analysis. The human PDAC 10-22 cell system is being used to discover effectors and networks whereby pluripotency can dominate over cancer and new features of human PDAC progression, which would be ultimately used for developing novel cancer therapies. Citation Format: Jungsun Kim, Greg Donahue, Wenli Yang, Kenneth S. Zaret.{Authors}. Stage-specific transcriptome analysis of human pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A91.

Single-cell pluripotency regulatory networks.

Pluripotent stem cells (PSCs) are a popular model system for investigating development, tissue regeneration, and repair. Although much is known about the molecular mechanisms that regulate the balance between self-renewal and lineage commitment in PSCs, the spatiotemporal integration of responsive signaling pathways with core transcriptional regulatory networks are complex and only partially understood. Moreover, measurements made on populations of cells reveal only average properties of the underlying regulatory networks, obscuring their fine detail. Here, we discuss the reconstruction of regulatory networks in individual cells using novel single-cell transcriptomics and proteomics, in order to expand our understanding of the molecular basis of pluripotency, including the role of cell-cell variability within PSC populations, and ways in which networks may be controlled in order to reliably manipulate cell behavior.

Role of Oct4 in the early embryo development.

Oct4 is a key component of the pluripotency regulatory network, and its reciprocal interaction with Cdx2 has been shown to be a determinant of either the self-renewal of embryonic stem cells (ESCs) or their differentiation into trophoblast. Oct4 of maternal origin is postulated to play critical role in defining totipotency and inducing pluripotency during embryonic development. However, the genetic elimination of maternal Oct4 using a Cre-lox approach in mouse revealed that the establishment of totipotency in maternal Oct4–depleted embryos was not affected, and that these embryos could complete full-term development without any obvious defect. These results indicate that Oct4 is not essential for the initiation of pluripotency, in contrast to its critical role in maintaining pluripotency. This conclusion is further supported by the formation of Oct4-GFP– and Nanog- expressing inner cell masses (ICMs) in embryos with complete inactivation of both maternal and zygotic Oct4 expression and the reprogramming of fibroblasts into fully pluripotent cells by Oct4-deficient oocytes.

A liaison between intrinsic and extrinsic regulators of pluripotency

A pluripotent cell state is regulated by complex transcription factor network under the control of extrinsic signals, so that undifferentiated cells can readily commit to any cell lineages in response to appropriate stimuli. A number of recent studies using pluripotent stem cells in vitro have shown that maintenance of pluripotency can be achieved with different combinations of extrinsic factors, yet by activating common intrinsic regulators. Two papers in this issue of The EMBO Journal identified Tfcp2l1 (also known as Crtr‐1 or Tcfcp2l1) as a factor on which activating and repressing stimuli for pluripotency converge. The results provide insight into how intrinsic pluripotency regulatory network is stably organized downstream of diverse inputs. Pluripotent stem cells in early embryos are transient cell populations that exist in the epiblast of the peri‐implantation embryos, but can be captured in vitro as embryonic stem cells (ESCs). ESCs self‐renew indefinitely while keeping the ability to differentiate into any cell lineages. Maintenance of self‐renewal in ESCs depends on self‐organizing transcriptional regulatory network, in which continuous inputs from extrinsic cues are required to sustain an intrinsic transcription factor (TF) circuitry. The most critical extrinsic regulators are leukaemia inhibitory factor (LIF) and its downstream effector Stat3. Although loss of zygotic Stat3 does not affect the embryonic development before gastrulation, a recent analysis of embryos lacking both maternal and zygotic Stat3 clearly revealed its indispensable role in the specification of the epiblast lineage around implantation (Do et al , 2013). However, consistent with other studies showing that lineage identities in early embryos …

Pou5f1 Transcription Factor Controls Zygotic Gene Activation In Vertebrates

The development of multicellular animals is initially controlled by maternal gene products deposited in the oocyte. During the maternal-to-zygotic transition, transcription of zygotic genes commences, and developmental control starts to be regulated by zygotic gene products. In Drosophila, the transcription factor Zelda specifically binds to promoters of the earliest zygotic genes and primes them for activation. It is unknown whether a similar regulation exists in other animals. We found that zebrafish Pou5f1, a homolog of the mammalian pluripotency transcription factor Oct4, occupies SOX-POU binding sites before the onset of zygotic transcription and activates the earliest zygotic genes. Our data position Pou5f1 and SOX-POU sites at the center of the zygotic gene activation network of vertebrates and provide a link between zygotic gene activation and pluripotency control.

WNT3 Is a Biomarker Capable of Predicting the Definitive Endoderm Differentiation Potential of hESCs

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However, the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here, we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition, manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally, analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively, our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs.

Learning the molecular mechanisms of the reprogramming factors: let's start from microRNAs

Induced reprogramming of somatic cells has had a great impact on stem cell research, and the reprogramming technologies have evolved from four transgenic factors (Oct4, Sox2, Klf4, and c-Myc; OSKM) to just a few microRNAs (mainly miR-290/302 seed family). Despite these advances, the molecular events occurring during various stages of reprogramming remain largely unknown. Here, we concisely review current knowledge of miRNA regulation from the initiation phase of OSKM-induced reprogramming, through the transitional stage, to final maturation. At the start of reprogramming, the microRNAs miR-21, miR-29a, let-7a, and miR-34 act as guards to secure the somatic identity and genomic integrity of the cell of origin. As reprogramming proceeds, miR-155, miR-10b, miR-205, and miR-429 modulate the epithelial-mesenchymal/mesenchymal-epithelial transition (EMT/MET), which is a critical step towards transformed pluripotent status. Finally, the pluripotency regulatory network is secured in the iPSCs and fine-tuned by a group of miRNAs belonging to the miR-290/302 seed family. Among the four reprogramming factors, c-Myc plays the dominant role in regulating the miRNAs under reprogramming-specific conditions. Accumulating evidence suggests that the reprogramming efficiency can be improved by either blocking barrier miRNAs or introducing helper miRNAs. Intriguingly, induced pluripotency can be obtained by introducing a single miR-302 cluster, although the supportive molecular mechanism is still lacking. In the near future, we may be able to realize the broad potential of miRNAs in the stem cell field, such as altering cell identities with high efficiency through the transient introduction of tissue-specific miRNAs.