ABSTRACT
Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.
Subject(s)
Circadian Clocks/genetics , Macrophages/physiology , Aging , Animals , Atherosclerosis/genetics , Cell Differentiation/genetics , Gene Expression Regulation/genetics , Immunity, Innate/genetics , Inflammation/genetics , Kruppel-Like Factor 4/genetics , Kruppel-Like Transcription Factors/genetics , Male , Mice , Mice, Inbred C57BL , Monocytes/physiology , Phagocytosis/geneticsABSTRACT
Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.
Subject(s)
Cellular Reprogramming , Transcription Factors/metabolism , Animals , Chromatin/metabolism , Fibroblasts/metabolism , Histone Code , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Mice , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Regulatory Elements, Transcriptional , SOXB1 Transcription Factors/metabolism , Silencer Elements, TranscriptionalABSTRACT
Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.
Subject(s)
Aging/genetics , Cellular Reprogramming , Epigenesis, Genetic , Metabolic Diseases/genetics , Transcription Factors/metabolism , Aging, Premature/genetics , Aging, Premature/metabolism , Animals , Diabetes Mellitus, Type 2/chemically induced , Diabetes Mellitus, Type 2/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Lamin Type A/genetics , Metabolic Diseases/metabolism , Metabolic Diseases/prevention & control , Mice , Models, Animal , Pancreas/metabolism , Sarcopenia/metabolismABSTRACT
Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5-7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.
Subject(s)
Epigenesis, Genetic , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Genome, Human , Germ Cells/metabolism , Animals , DNA Methylation , Embryo, Mammalian/metabolism , Female , Humans , Kruppel-Like Factor 4 , Male , Mice , Promoter Regions, Genetic , RetroelementsABSTRACT
Pioneer transcription factors (TFs) access silent chromatin and initiate cell-fate changes, using diverse types of DNA binding domains (DBDs). FoxA, the paradigm pioneer TF, has a winged helix DBD that resembles linker histone and thereby binds its target sites on nucleosomes and in compacted chromatin. Herein, we compare the nucleosome and chromatin targeting activities of Oct4 (POU DBD), Sox2 (HMG box DBD), Klf4 (zinc finger DBD), and c-Myc (bHLH DBD), which together reprogram somatic cells to pluripotency. Purified Oct4, Sox2, and Klf4 proteins can bind nucleosomes in vitro, and in vivo they preferentially target silent sites enriched for nucleosomes. Pioneer activity relates simply to the ability of a given DBD to target partial motifs displayed on the nucleosome surface. Such partial motif recognition can occur by coordinate binding between factors. Our findings provide insight into how pioneer factors can target naive chromatin sites.
Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/cytology , Nucleosomes/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Cell Dedifferentiation , DNA/metabolism , Fibroblasts/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Models, Molecular , Nucleotide Motifs , Octamer Transcription Factor-3/metabolism , Protein Structure, Tertiary , Sequence Alignment , Transcription Factors/chemistry , Transcription Factors/classificationABSTRACT
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Subject(s)
CRISPR-Cas Systems , Hepatocytes , Liver Neoplasms , Liver , Neoplasm Metastasis , Nerve Tissue Proteins , Animals , Female , Humans , Male , Mice , Cell Line, Tumor , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , CRISPR-Cas Systems/genetics , Disease Models, Animal , DNA Transposable Elements , Fluorescence , Hepatocytes/metabolism , Hepatocytes/cytology , Hepatocytes/pathology , Kruppel-Like Factor 4/metabolism , Liver/cytology , Liver/metabolism , Liver/pathology , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/prevention & control , Liver Neoplasms/secondary , Melanoma/metabolism , Melanoma/pathology , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/pathology , Neoplasm Metastasis/prevention & control , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Semaphorins/antagonists & inhibitors , Semaphorins/metabolismABSTRACT
Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.
Subject(s)
Leukemia, Promyelocytic, Acute , Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Humans , Kruppel-Like Factor 4 , Leukemia, Promyelocytic, Acute/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Transcription Factors/metabolism , Tretinoin/pharmacologyABSTRACT
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
Subject(s)
Homeodomain Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Pluripotent Stem Cells/metabolism , Animals , Cytological Techniques , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Humans , Kruppel-Like Factor 4 , Mice , Mitochondria/metabolism , Nanog Homeobox Protein , Pluripotent Stem Cells/cytology , Transcription Factors/metabolism , TranscriptomeABSTRACT
The past 10 years have seen great advances in our ability to manipulate cell fate, including the induction of pluripotency in vitro to generate induced pluripotent stem cells (iPSCs). This process proved to be remarkably simple from a technical perspective, only needing the host cell and a defined cocktail of transcription factors, with four factors - octamer-binding protein 3/4 (OCT3/4), SOX2, Krüppel-like factor 4 (KLF4) and MYC (collectively referred to as OSKM) - initially used. The mechanisms underlying transcription factor-mediated reprogramming are still poorly understood; however, several mechanistic insights have recently been obtained. Recent years have also brought significant progress in increasing the efficiency of this technique, making it more amenable to applications in the fields of regenerative medicine, disease modelling and drug discovery.
Subject(s)
Cellular Reprogramming Techniques/methods , Cellular Reprogramming , Transcription Factors , Animals , Cellular Reprogramming Techniques/history , History, 21st Century , Humans , Kruppel-Like Factor 4 , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
Germline determinants of gene expression in tumors are infrequently studied due to the complexity of transcript regulation caused by somatically acquired alterations. We performed expression quantitative trait locus (eQTL)-based analyses using the multi-level information provided in The Cancer Genome Atlas (TCGA). Of the factors we measured, cis-acting eQTLs accounted for 1.2% of the total variation of tumor gene expression, while somatic copy-number alteration and CpG methylation accounted for 7.3% and 3.3%, respectively. eQTL analyses of 15 previously reported breast cancer risk loci resulted in the discovery of three variants that are significantly associated with transcript levels (false discovery rate [FDR] < 0.1). Our trans-based analysis identified an additional three risk loci to act through ESR1, MYC, and KLF4. These findings provide a more comprehensive picture of gene expression determinants in breast cancer as well as insights into the underlying biology of breast cancer risk loci.
Subject(s)
Breast Neoplasms/genetics , Genetic Predisposition to Disease , Genome-Wide Association Study , Quantitative Trait Loci , Cell Line, Tumor , Gene Expression Profiling , Humans , Kruppel-Like Factor 4ABSTRACT
Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.
Subject(s)
5-Methylcytosine/analysis , Cytosine/analogs & derivatives , DNA Methylation , 5-Methylcytosine/metabolism , Animals , Brain/cytology , Brain/metabolism , Cytosine/analysis , Cytosine/metabolism , DNA Glycosylases/metabolism , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/metabolism , Kruppel-Like Factor 4 , Mass Spectrometry , Mice , Oxidation-Reduction , Proto-Oncogene Proteins/metabolism , Regulatory Factor X Transcription Factors , Stem Cells/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.
Subject(s)
Cell Differentiation/genetics , Epidermal Cells/cytology , Epidermis/embryology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Animals , Embryo, Mammalian , Gene Deletion , Gene Expression Regulation, Developmental , Genes, Lethal/genetics , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Inbred C57BL , Mutation , Nuclear Receptor Co-Repressor 1/genetics , Nuclear Receptor Co-Repressor 1/metabolism , Nuclear Receptor Co-Repressor 2/genetics , Nuclear Receptor Co-Repressor 2/metabolism , Protein Interaction Domains and Motifs/genetics , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolismABSTRACT
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
The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hr of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include an unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed.
Subject(s)
Cellular Reprogramming , Fibroblasts/metabolism , Genome, Human , Pluripotent Stem Cells/metabolism , Chromatin/metabolism , Embryonic Stem Cells/metabolism , Fibroblasts/cytology , Gene Expression , Histone Code , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Models, Molecular , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , Proto-Oncogene Proteins c-myc/metabolism , SOXB1 Transcription Factors/metabolismABSTRACT
Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripotent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved exceedingly inefficient. We discovered a striking difference in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogramming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain- and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables efficient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory pathways are required for efficient nuclear reprogramming in the induction of pluripotency.
Subject(s)
Cell-Penetrating Peptides/metabolism , Cellular Reprogramming , Immunity, Innate , Induced Pluripotent Stem Cells/metabolism , Signal Transduction , Cell Line , Fibroblasts/metabolism , Humans , Inflammation/metabolism , Kruppel-Like Factor 4 , NF-kappa B/metabolism , Octamer Transcription Factor-3/metabolism , Retroviridae/metabolism , Toll-Like Receptor 3/metabolismABSTRACT
During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.
Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/metabolism , Single-Cell Analysis , Transcriptome , Animals , Cell Line , Embryo, Mammalian/cytology , Embryonic Stem Cells , Fibroblasts/cytology , Fibroblasts/metabolism , Genetic Markers , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Factor 4 , Mice , Microfluidic Analytical Techniques , SOXB1 Transcription Factors/metabolism , Transcription Factors/metabolismABSTRACT
Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.
Subject(s)
Class I Phosphatidylinositol 3-Kinases/genetics , Hemangioma, Cavernous, Central Nervous System/genetics , Hemangioma, Cavernous, Central Nervous System/pathology , Mutation , Neoplasms/genetics , Animals , Animals, Newborn , Class I Phosphatidylinositol 3-Kinases/metabolism , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gain of Function Mutation , Hemangioma, Cavernous, Central Nervous System/blood supply , Hemangioma, Cavernous, Central Nervous System/metabolism , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Loss of Function Mutation , MAP Kinase Kinase Kinase 3/metabolism , Male , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Neoplasms/blood supply , Neoplasms/pathology , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/metabolismABSTRACT
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a highly heterogeneous process. Here we report the cell fate continuum during somatic cell reprogramming at single-cell resolution. We first develop SOT to analyze cell fate continuum from Oct4/Sox2/Klf4- or OSK-mediated reprogramming and show that cells bifurcate into two categories, reprogramming potential (RP) or non-reprogramming (NR). We further show that Klf4 contributes to Cd34+/Fxyd5+/Psca+ keratinocyte-like NR fate and that IFN-γ impedes the final transition to chimera-competent pluripotency along the RP cells. We analyze more than 150,000 single cells from both OSK and chemical reprograming and identify additional NR/RP bifurcation points. Our work reveals a generic bifurcation model for cell fate decisions during somatic cell reprogramming that may be applicable to other systems and inspire further improvements for reprogramming.
Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Cellular Reprogramming Techniques , Cellular Reprogramming/genetics , Induced Pluripotent Stem Cells/physiology , Mouse Embryonic Stem Cells/physiology , Sequence Analysis, RNA , Single-Cell Analysis , Animals , Female , Gene Expression Regulation, Developmental , Induced Pluripotent Stem Cells/metabolism , Interferon-gamma/genetics , Interferon-gamma/metabolism , Kruppel-Like Factor 4 , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Mouse Embryonic Stem Cells/metabolism , Phenotype , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.
Subject(s)
Cell Differentiation/genetics , Gene Expression Regulation, Developmental/genetics , Kruppel-Like Transcription Factors/metabolism , Transcription Factors/metabolism , Active Transport, Cell Nucleus , Animals , Cell Line , Embryonic Stem Cells , HEK293 Cells , Humans , Kruppel-Like Factor 4 , Mice , Mutation/genetics , Protein Domains , Protein Stability , Proteolysis , RNA Polymerase II/metabolism , Signal Transduction , UbiquitinationABSTRACT
The discovery of somatic cell nuclear transfer proved that somatic cells can carry the same genetic code as the zygote, and that activating parts of this code are sufficient to reprogram the cell to an early developmental state. The discovery of induced pluripotent stem cells (iPSCs) nearly half a century later provided a molecular mechanism for the reprogramming. The initial creation of iPSCs was accomplished by the ectopic expression of four specific genes (OCT4, KLF4, SOX2, and c-Myc; OSKM). iPSCs have since been acquired from a wide range of cell types and a wide range of species, suggesting a universal molecular mechanism. Furthermore, cells have been reprogrammed to iPSCs using a myriad of methods, although OSKM remains the gold standard. The sources for iPSCs are abundant compared with those for other pluripotent stem cells; thus the use of iPSCs to model the development of tissues, organs, and other systems of the body is increasing. iPSCs also, through the reprogramming of patient samples, are being used to model diseases. Moreover, in the 10 years since the first report, human iPSCs are already the basis for new cell therapies and drug discovery that have reached clinical application. In this review, we examine the generation of iPSCs and their application to disease and development.