ABSTRACT
Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
Subject(s)
Cellular Reprogramming , Cytological Techniques/methods , Induced Pluripotent Stem Cells/cytology , Animals , Genome-Wide Association Study , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Mice , Transcription Factors/metabolismABSTRACT
Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1-4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.
Subject(s)
Blastocyst/cytology , Blastocyst/metabolism , Cell Culture Techniques , Cellular Reprogramming , Fibroblasts/cytology , Models, Biological , Transcriptome , Female , Fibroblasts/metabolism , Humans , In Vitro Techniques , Single-Cell Analysis , Stem Cells/cytology , Stem Cells/metabolism , Trophoblasts/cytologyABSTRACT
Nuclear transporter Importin (Imp, Ipo) 13 is known to transport various mammalian cargoes into/out of the nucleus, but its role in directing cell-fate is unclear. Here we examine the role of Imp13 in the maintenance of pluripotency and differentiation of embryonic stem cells (ESCs) for the first time, using an embryonic body (EB)-based model. When induced to differentiate, Ipo13-/- ESCs displayed slow proliferation, reduced EB size, and lower expression of the proliferation marker KI67, concomitant with an increase in the number of TUNEL+ nuclei compared to wildtype ESCs. At days 5 and 10 of differentiation, Ipo13-/- EBs also showed enhanced loss of the pluripotency transcript OCT3/4, and barely detectable clusters of OCT3/4 positive cells. Day 5 Ipo13-/- EBs further exhibited reduced levels of the mesodermal markers Brachyury and Mixl1, correlating with reduced numbers of haemoglobinised cells generated. Our findings suggest that Imp13 is critical to ESC survival as well as early post-gastrulation differentiation.
Subject(s)
Embryonic Stem Cells/cytology , Karyopherins/physiology , Animals , Apoptosis , Cell Differentiation , Cell Proliferation , Cell Survival , Embryoid Bodies/metabolism , Gene Knockout Techniques , Karyopherins/genetics , Mesoderm/metabolism , Mice , Octamer Transcription Factor-3/metabolismABSTRACT
The canonical Wnt/ß-catenin pathway is crucial for early embryonic patterning, tissue homeostasis, and regeneration. While canonical Wnt/ß-catenin stimulation has been used extensively to modulate pluripotency and differentiation of pluripotent stem cells (PSCs), the mechanism of these two seemingly opposing roles has not been fully characterized and is currently largely attributed to activation of nuclear Wnt target genes. Here, we show that low levels of Wnt stimulation via ectopic expression of Wnt1 or administration of glycogen synthase kinase-3 inhibitor CHIR99021 significantly increases PSC differentiation into neurons, cardiomyocytes and early endodermal intermediates. Our data indicate that enhanced differentiation outcomes are not mediated through activation of traditional Wnt target genes but by ß-catenin's secondary role as a binding partner of membrane bound cadherins ultimately leading to the activation of developmental genes. In summary, fine-tuning of Wnt signaling to subthreshold levels for detectable nuclear ß-catenin function appears to act as a switch to enhance differentiation of PSCs into multiple lineages. Our observations highlight a mechanism by which Wnt/ß-catenin signaling can achieve dosage dependent dual roles in regulating self-renewal and differentiation. Stem Cells 2018;36:822-833.
Subject(s)
Pluripotent Stem Cells/metabolism , Wnt Signaling Pathway/genetics , beta Catenin/genetics , Animals , Cell Differentiation , Humans , Mice , Signal TransductionABSTRACT
Cellular reprogramming refers to the conversion of one cell type into another by altering its epigenetic marks. This can be achieved by three different methods: somatic cell nuclear transfer, cell fusion and transcription factor (TF)-mediated reprogramming. TF-mediated reprogramming can occur through several means, either reverting backwards to a pluripotent state before redifferentiating to a new cell type (otherwise known as induced pluripotency), by transdifferentiating directly into a new cell type (bypassing the intermediate pluripotent stage), or, by using the induced pluripotency pathway without reaching the pluripotent state. The possibility of reprogramming any cell type of interest not only sheds new insights on cellular plasticity, but also provides a novel use of this technology across several platforms, most notably in cellular replacement therapies, disease modelling and drug screening. This review will focus on the different ways of implementing TF-mediated reprogramming, their associated epigenetic changes and its therapeutic potential.
Subject(s)
Cell Transdifferentiation/genetics , Cellular Reprogramming/genetics , Epigenesis, Genetic , Induced Pluripotent Stem Cells/cytology , Regenerative Medicine , Transcription Factors/metabolism , Animals , Biological Therapy , HumansABSTRACT
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Subject(s)
Cell Separation/methods , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/metabolism , Myoblasts, Cardiac/cytology , Myocytes, Cardiac/cytology , Transcription Factors/metabolism , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Biomarkers/analysis , Cell Differentiation , Gene Expression Profiling , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Humans , Myoblasts, Cardiac/metabolism , Myocytes, Cardiac/metabolism , Polymerase Chain Reaction , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Transcription Factors/genetics , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method's utility in stem cell disease modeling.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , CRISPR-Cas Systems/genetics , DNA/metabolism , Cell Line , Cloning, Molecular , Gene Editing/methodsABSTRACT
We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.
Subject(s)
Cell Lineage/genetics , Cell Tracking/methods , Embryonic Stem Cells/metabolism , Nuclear Proteins/genetics , Prosencephalon/embryology , Transcription Factors/genetics , Animals , Animals, Newborn , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Embryonic Stem Cells/cytology , Flow Cytometry/methods , Genes, Reporter , Humans , Mice , Mice, Transgenic , Molecular Targeted Therapy/methods , Neurogenesis/genetics , Neurogenesis/physiology , Nuclear Proteins/metabolism , Prosencephalon/cytology , Prosencephalon/physiology , Thyroid Nuclear Factor 1 , Transcription Factors/metabolismABSTRACT
Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Mice , Animals , Retroelements/genetics , DNA Transposable Elements/genetics , Mutation , Long Interspersed Nucleotide Elements/geneticsABSTRACT
The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1(C) ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)(+) cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1(C) ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1(C) EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation.
Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endoderm/metabolism , Homeodomain Proteins/physiology , Mesoderm/cytology , Mesoderm/metabolism , Animals , BALB 3T3 Cells , Blotting, Western , Cadherins/genetics , Cadherins/metabolism , Cell Differentiation/genetics , Cell Line , Electrophoretic Mobility Shift Assay , Endoderm/cytology , Flow Cytometry , HMGB Proteins/genetics , HMGB Proteins/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , Hepatocyte Nuclear Factor 3-beta/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunohistochemistry , Mice , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolismABSTRACT
Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.
Subject(s)
Genetic Loci , Human Embryonic Stem Cells/metabolism , Multiprotein Complexes/metabolism , Nanog Homeobox Protein/metabolism , Co-Repressor Proteins/metabolism , Human Embryonic Stem Cells/cytology , HumansABSTRACT
Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.
Subject(s)
CpG Islands , DNA Methylation , DNA, Neoplasm/metabolism , Histones/metabolism , Neoplasms/metabolism , 5-Methylcytosine/analogs & derivatives , 5-Methylcytosine/metabolism , Animals , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Gene Expression Regulation, Neoplastic , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Humans , Male , Methylation , Mice, Inbred C57BL , Mice, Knockout , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/genetics , Promoter Regions, GeneticABSTRACT
Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) induces changes in genome architecture reflective of the embryonic stem cell (ESC) state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of the process. Here, we characterize the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of intermediates poised to become iPSCs. Widespread reconfiguration of chromatin states and transcription factor (TF) occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain their original chromatin states. A second wave of reconfiguration occurs just prior to pluripotency acquisition, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of reprogramming-associated molecular changes broadens our understanding of this process and sheds light on how TFs access and change the chromatin during cell-fate transitions.
Subject(s)
Cellular Reprogramming , Chromatin/metabolism , Induced Pluripotent Stem Cells/metabolism , Transcription Factors/metabolism , Animals , Cellular Reprogramming/genetics , Chromatin/genetics , Female , Induced Pluripotent Stem Cells/cytology , Mice , Mice, Inbred NOD , Mice, SCID , Transcription Factors/geneticsABSTRACT
Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources.
Subject(s)
Fibroblasts/cytology , Neutrophils/cytology , Animals , Cellular Reprogramming/physiology , Early Growth Response Protein 1/metabolism , Fibroblasts/physiology , Flow Cytometry , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Keratinocytes/cytology , Keratinocytes/physiology , Neutrophils/physiology , Octamer Transcription Factor-3/metabolismABSTRACT
UNLABELLED: Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. SIGNIFICANCE: This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/virology , Host-Pathogen Interactions , Nuclear Proteins , Picornaviridae Infections/mortality , Respiratory Mucosa/virology , Rhinovirus/physiology , Transcription Factors , Animals , Cell Line , Embryonic Stem Cells/metabolism , Humans , Mice , Mice, Nude , Picornaviridae Infections/pathology , Respiratory Mucosa/metabolism , Thyroid Nuclear Factor 1ABSTRACT
The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34(+)KDR(+) endothelial progenitor cells after 6days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.
Subject(s)
Culture Media/pharmacology , Embryonic Stem Cells/drug effects , Endothelial Cells/cytology , Lysophospholipids/pharmacology , Nitric Oxide Synthase Type III/analysis , Platelet Activating Factor/pharmacology , Antigens, CD34/metabolism , Cell Differentiation/drug effects , Cell Line , Collagen/chemistry , Drug Combinations , Embryonic Stem Cells/cytology , Endothelial Cells/metabolism , Gene Expression Profiling , Human Umbilical Vein Endothelial Cells , Humans , Laminin/chemistry , Nitric Oxide Synthase Type III/metabolism , Proteoglycans/chemistry , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
To develop methods for the generation of insulin-producing ß-cells for the treatment of diabetes, we have used GFP-tagged embryonic stem cells (ESCs) to elucidate the process of pancreas development. Using the reporter Pdx1(GFP/w) ESC line, we have previously described a serum-free differentiation protocol in which Pdx1-GFP(+) cells formed GFP bright (GFP(br)) epithelial buds that resembled those present in the developing mouse pancreas. In this study we extend these findings to demonstrate that these cells can undergo a process of branching morphogenesis, similar to that seen during pancreatic development of the mid-gestation embryo. These partially disaggregated embryoid bodies containing GFP(br) buds initially form epithelial ring-like structures when cultured in Matrigel. After several days in culture, these rings undergo a process of proliferation and form a ramified network of epithelial branches. Comparative analysis of explanted dissociated pancreatic buds from E13.5 Pdx1(GFP/w) embryos and ESC-derived GFP(br) buds reveal a similar process of proliferation and branching, with both embryonic Pdx1(GFP/w) branching pancreatic epithelium and ESC-derived GFP(br) branching organoids expressing markers representing epithelial (EpCAM and E-Cadherin), ductal (Mucin1), exocrine (Amylase and Carboxypeptidase 1A), and endocrine cell types (Glucagon and Somatostatin). ESC-derived branching structures also expressed a suite of genes indicative of ongoing pancreatic differentiation, paralleling gene expression within similar structures derived from the E13.5 fetal pancreas. In summary, differentiating mouse ESCs can generate pancreatic material that has significant similarity to the fetal pancreatic anlagen, providing an in vitro platform for investigating the cellular and molecular mechanisms underpinning pancreatic development.
Subject(s)
Embryoid Bodies/physiology , Embryonic Development , Organogenesis , Pancreas/embryology , Animals , Cell Differentiation , Cells, Cultured , Coculture Techniques , Embryoid Bodies/metabolism , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Endoderm/cytology , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence , Pancreas/cytology , Pancreas/metabolism , Real-Time Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tissue Culture Techniques , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in the Pdgfrα and Flk1 promoters and chromatin immunoprecipitation assays confirmed Mixl1 occupancy of these promoters in vivo. Furthermore, Mixl1 transactivated the Pdgfrα and Flk1 promoters through ATTA sequences in a DNA binding dependent manner. These data support the hypothesis that Mixl1 directly regulates Pdgfrα and Flk1 gene expression and strengthens the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.
Subject(s)
Cell Differentiation/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Homeodomain Proteins/metabolism , Receptor, Platelet-Derived Growth Factor alpha/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Endoderm/cytology , Endoderm/metabolism , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Developmental , Homeodomain Proteins/chemistry , Mesoderm/cytology , Mesoderm/metabolism , Mice , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Transcriptional Activation/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.
Subject(s)
Fetal Proteins/metabolism , Gene Expression Regulation , Homeodomain Proteins/metabolism , T-Box Domain Proteins/metabolism , Animals , Cell Differentiation , Cell Line, Tumor , Cell Nucleus/metabolism , Fibroblasts/metabolism , Fluorescent Antibody Technique, Indirect , HEK293 Cells , Humans , Mice , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Interaction Mapping , Transcription, GeneticABSTRACT
Slain1 was originally identified as a novel stem cell-associated gene in transcriptional profiling experiments comparing mouse and human embryonic stem cells (ESCs) and their immediate differentiated progeny. In order to obtain further insight into the potential function of Slain1, we examined the expression of beta-galactosidase in a gene-trap mouse line in which a beta-geo reporter gene was inserted into the second intron of Slain1. In early stage embryos (E7.5), the Slain1-betageo fusion protein was expressed within the entire epiblast, but by E9.5 became restricted to the developing nervous system and gastrointestinal tract. In later stage embryos (E11.5 - E13.5), expression was predominantly within the developing nervous system. Lower level expression was also observed in the developing limb buds, in the condensing mesenchyme, along the apical epidermal ridge and, at later stages, within the digital zones. These observations suggest that Slain1 may play a role in the development of the nervous system, as well as in the morphogenesis of several embryonic structures.