The SMAD2/3 interactome reveals that TGFß controls m6A mRNA methylation in pluripotency.

The TGFß pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA. We show that SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFß signalling could have far-reaching implications in many other cell types and in diseases such as cancer.

Cell cycle regulators control mesoderm specification in human pluripotent stem cells.

The mesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys, and the cardiovascular system originate. Understanding the mechanisms that control mesoderm specification could inform many applications, including the development of regenerative medicine therapies to manage diseases affecting these tissues. Here, we used human pluripotent stem cells to investigate the role of cell cycle in mesoderm formation. To this end, using small molecules or conditional gene knockdown, we inhibited proteins controlling G1 and G2/M cell cycle phases during the differentiation of human pluripotent stem cells into lateral plate, cardiac, and presomitic mesoderm. These loss-of-function experiments revealed that regulators of the G1 phase, such as cyclin-dependent kinases and pRb (retinoblastoma protein), are necessary for efficient mesoderm formation in a context-dependent manner. Further investigations disclosed that inhibition of the G2/M regulator cyclin-dependent kinase 1 decreases BMP (bone morphogenetic protein) signaling activity specifically during lateral plate mesoderm formation while reducing fibroblast growth factor/extracellular signaling-regulated kinase 1/2 activity in all mesoderm subtypes. Taken together, our findings reveal that cell cycle regulators direct mesoderm formation by controlling the activity of key developmental pathways.

Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle.

The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal, and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. J. Cell. Physiol. 232: 1254-1257, 2017. © 2016 Wiley Periodicals, Inc.

Lineage-Specific Early Differentiation of Human Embryonic Stem Cells Requires a G2 Cell Cycle Pause.

Human embryonic stem cells (hESCs) have an abbreviated G1 phase of the cell cycle that allows rapid proliferation and maintenance of pluripotency. Lengthening of G1 corresponds to loss of pluripotency during differentiation. However, precise mechanisms that link alterations in the cell cycle and early differentiation remain to be defined. We investigated initial stages of mesendodermal lineage commitment in hESCs, and observed a cell cycle pause. Transcriptome profiling identified several genes with known roles in regulation of the G2/M transition that were differentially expressed early during lineage commitment. WEE1 kinase, which blocks entry into mitosis by phosphorylating CDK1 at Y15, was the most highly expressed of these genes. Inhibition of CDK1 phosphorylation by a specific inhibitor of WEE1 restored cell cycle progression by preventing the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. Our findings identify a novel G2 cell cycle pause that is required for endodermal differentiation and provide important new mechanistic insights into early events of lineage commitment. Stem Cells 2016;34:1765-1775.

Expression of Ribosomal RNA and Protein Genes in Human Embryonic Stem Cells Is Associated With the Activating H3K4me3 Histone Mark.

Embryonic stem cells (ESCs) exhibit unrestricted and indefinite, but stringently controlled, proliferation, and can differentiate into any lineage in the body. In the current study, we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs, we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines, a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches, we discovered that, RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1, M, and G2 phases of the cell cycle. Interestingly, the rDNA repeats are marked by the activating H3K4me3 only in the M phase, and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc, a known regulator of cell growth and proliferation, occupies both the rRNA genes and RPGs. Functionally, down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together, our results show that expression of rRNA, which is regulated by the Myc pluripotency transcription factor, and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013, 2016. © 2016 Wiley Periodicals, Inc.

A functional N-terminal domain in C/EBPß-LAP* is required for interacting with SWI/SNF and to repress Ric-8B gene transcription in osteoblasts.

The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPß play critical roles in osteoblastic cells as they jointly control transcription of a number of bone-related target genes. The largest C/EBPß isoform, LAP*, possesses a short additional N-terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N-terminus in C/EBPß-LAP* for binding SWI/SNF and for recruiting this complex to the Ric-8B gene to mediate transcriptional repression. We find that both C/EBPß-LAP* and SWI/SNF simultaneously bind to the Ric-8B promoter in differentiating osteoblasts that repress Ric-8B expression. This decreased expression of Ric-8B is not accompanied by significant changes in histone acetylation at the Ric-8B gene promoter sequence. A single aminoacid change at the C/EBPß-LAP* N-terminus (R3L) that inhibits C/EBPß-LAP*-SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric-8B promoter as well as C/EBPß-LAP*-dependent repression of the Ric-8B gene. Inducible expression of the C/EBPß-LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPß-LAP*-target promoters and competes with the endogenous C/EBPß factor. Together our results indicate that a functional N-terminus in C/EBPß-LAP* is required for interacting with SWI/SNF and for Ric-8B gene repression in osteoblasts.

The dynamic architectural and epigenetic nuclear landscape: developing the genomic almanac of biology and disease.

Compaction of the eukaryotic genome into the confined space of the cell nucleus must occur faithfully throughout each cell cycle to retain gene expression fidelity. For decades, experimental limitations to study the structural organization of the interphase nucleus restricted our understanding of its contributions towards gene regulation and disease. However, within the past few years, our capability to visualize chromosomes in vivo with sophisticated fluorescence microscopy, and to characterize chromosomal regulatory environments via massively parallel sequencing methodologies have drastically changed how we currently understand epigenetic gene control within the context of three-dimensional nuclear structure. The rapid rate at which information on nuclear structure is unfolding brings challenges to compare and contrast recent observations with historic findings. In this review, we discuss experimental breakthroughs that have influenced how we understand and explore the dynamic structure and function of the nucleus, and how we can incorporate historical perspectives with insights acquired from the ever-evolving advances in molecular biology and pathology.

The abbreviated pluripotent cell cycle.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory, and structural. The primary temporal context that the pluripotent self-renewal cell cycle of hESCs is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the embryonic stem cell (ESC) cell cycle. This supports the requirements of pluripotent cells to self-propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated ESC cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle.

Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation.

Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.

Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation.

BACKGROUND: Haematopoietic stem cells (HSCs) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. RESULTS: In this study, we take advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrate that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. CONCLUSION: We propose a direct link between the molecular machineries that control cell cycle progression and EHT.

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation.

The ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a "differentiation checkpoint" in early endoderm differentiation that alters cell fate in response to DNA damage.

Modeling Disease with Human Inducible Pluripotent Stem Cells.

Understanding the physiopathology of disease remains an essential step in developing novel therapeutics. Although animal models have certainly contributed to advancing this enterprise, their limitation in modeling all the aspects of complex human disorders is one of the major challenges faced by the biomedical research field. Human induced pluripotent stem cells (hiPSCs) derived from patients represent a great opportunity to overcome this deficiency because these cells cover the genetic diversity needed to fully model human diseases. Here, we provide an overview of the history of hiPSC technology and discuss common challenges and approaches that we and others have faced when using hiPSCs to model disease. Our emphasis is on liver disease, and consequently, we review the progress made using this technology to produce functional liver cells in vitro and how these systems are being used to recapitulate a diversity of developmental, metabolic, genetic, and infectious liver disorders.

Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics.

Cell cycle progression and cell fate decisions are closely linked in human pluripotent stem cells (hPSCs). However, the study of these interplays at the molecular level remains challenging due to the lack of efficient methods allowing cell cycle synchronization of large quantities of cells. Here, we screened inhibitors of cell cycle progression and identified nocodazole as the most efficient small molecule to synchronize hPSCs in the G2/M phase. Following nocodazole treatment, hPSCs remain pluripotent, retain a normal karyotype and can successfully differentiate into the three germ layers and functional cell types. Moreover, genome-wide transcriptomic analyses on single cells synchronized for their cell cycle and differentiated toward the endoderm lineage validated our findings and showed that nocodazole treatment has no effect on gene expression during the differentiation process. Thus, our synchronization method provides a robust approach to study cell cycle mechanisms in hPSCs.

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency.

Bookmarking target genes in mitosis: a shared epigenetic trait of phenotypic transcription factors and oncogenes?

The regulatory information for phenotype, proliferation, and growth of normal and tumor cells must be maintained through genome replication in the S phase and cell division during mitosis. Epigenetic mechanisms that include DNA methylation, posttranslational modifications of histones, selective utilization of histone variants, and inheritable RNA molecules play pivotal roles in maintaining cellular identity through mitotic divisions. Recent studies demonstrate that mitotic occupancy of genes, which are determinants of cell fate, growth, and proliferation, by lineage-restricted transcription factors is a key epigenetic mechanism for retention and transmission of cellular expression memory. Evidence is emerging for the presence of distinct transcriptional regulatory microenvironments in mitotic chromosomes in which the genes bookmarked for reactivation postmitotically reside. Importantly, some oncoproteins are present in mitotic microenvironments where they occupy target genes during mitosis and may contribute to perpetuating the transformed phenotype. We discuss emerging regulatory implications of epigenetically bookmarking genes during mitosis for physiologic control as well as for the onset and progression of cancer.

The architectural organization of human stem cell cycle regulatory machinery.

Two striking features of human embryonic stem cells that support biological activity are an abbreviated cell cycle and reduced complexity to nuclear organization. The potential implications for rapid proliferation of human embryonic stem cells within the context of sustaining pluripotency, suppressing phenotypic gene expression and linkage to simplicity in the architectural compartmentalization of regulatory machinery in nuclear microenvironments is explored. Characterization of the molecular and architectural commitment steps that license human embryonic stem cells to initiate histone gene expression is providing understanding of the principal regulatory mechanisms that control the G1/S phase transition in primitive pluripotent cells. From both fundamental regulatory and clinical perspectives, further understanding of the pluripotent cell cycle in relation to compartmentalization of regulatory machinery in nuclear microenvironments is relevant to applications of stem cells for regenerative medicine and new dimensions to therapy where traditional drug discovery strategies have been minimally effective.

The Ric-8B gene is highly expressed in proliferating preosteoblastic cells and downregulated during osteoblast differentiation in a SWI/SNF- and C/EBPbeta-mediated manner.

The Ric-8 gene encodes a guanine exchange factor (GEF) that modulates G protein-mediated signaling, exhibiting a relevant role during regulation of cell division. In mammals, two Ric-8 homologues have been reported (Ric-8A and Ric-8B), and recent studies indicate equivalent roles for each protein. Here, we show that the Ric-8B gene is negatively regulated during osteoblast differentiation by the transcription factor C/EBPß. Only the larger C/EBPß isoform (C/EBPß-LAP*) downregulates Ric-8B gene promoter activity in osteoblastic cells. Accordingly, knockdown of C/EBPß expression by small intefering RNA in osteoblastic cells results in a significant increase of Ric-8B gene expression. Transient overexpression of Brg1 or Brm, the catalytic subunits of the SWI/SNF chromatin-remodeling complex, inhibits Ric-8B promoter activity. Also, the presence of inactive SWI/SNF complexes in osteoblastic cells results in increased endogenous Ric-8B transcription, indicating that SWI/SNF activity negatively regulates Ric-8B expression. During osteoblast differentiation, Ric-8B gene repression is accompanied by changes in nucleosome placement at the proximal Ric-8B gene promoter and reduced accessibility to regulatory sequences.

Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.

A Gbetagamma stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation.

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.

Peroxisomal proliferation protects from beta-amyloid neurodegeneration.

Alzheimer disease is a neurodegenerative process that leads to severe cognitive impairment as a consequence of selective death of neuronal populations. The molecular pathogenesis of Alzheimer disease involves the participation of the beta-amyloid peptide (Abeta) and oxidative stress. We report here that peroxisomal proliferation attenuated Abeta-dependent toxicity in hippocampal neurons. Pretreatment with Wy-14.463 (Wy), a peroxisome proliferator, prevent the neuronal cell death and neuritic network loss induced by the Abeta peptide. Moreover, the hippocampal neurons treated with this compound, showed an increase in the number of peroxisomes, with a concomitant increase in catalase activity. Additionally, we evaluate the Wy protective effect on beta-catenin levels, production of intracellular reactive oxygen species, cytoplasmic calcium uptake, and mitochondrial potential in hippocampal neurons exposed to H(2) O(2) and Abeta peptide. Results show that the peroxisomal proliferation prevents beta-catenin degradation, reactive oxygen species production, cytoplasmic calcium increase, and changes in mitochondrial viability. Our data suggest, for the first time, a direct link between peroxisomal proliferation and neuroprotection from Abeta-induced degenerative changes.