ABSTRACT
Huntington's disease (HD) is a neurodegenerative disorder caused by CAG-repeat expansions in the huntingtin (HTT) gene. The resulting mutant HTT (mHTT) protein induces toxicity and cell death via multiple mechanisms and no effective therapy is available. Here, we employ a genome-wide screening in pluripotent mouse embryonic stem cells (ESCs) to identify suppressors of mHTT toxicity. Among the identified suppressors, linked to HD-associated processes, we focus on Metal response element binding transcription factor 1 (Mtf1). Forced expression of Mtf1 counteracts cell death and oxidative stress caused by mHTT in mouse ESCs and in human neuronal precursor cells. In zebrafish, Mtf1 reduces malformations and apoptosis induced by mHTT. In R6/2 mice, Mtf1 ablates motor defects and reduces mHTT aggregates and oxidative stress. Our screening strategy enables a quick in vitro identification of promising suppressor genes and their validation in vivo, and it can be applied to other monogenic diseases.
Subject(s)
Huntington Disease , Neurodegenerative Diseases , Mice , Animals , Humans , Disease Models, Animal , Zebrafish/genetics , Zebrafish/metabolism , Huntington Disease/metabolism , Neurons/metabolism , Neurodegenerative Diseases/metabolism , Huntingtin Protein/genetics , Huntingtin Protein/metabolismABSTRACT
In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineages, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibition combined with LIF stimulation is used to chemically reset conventional to naive PSCs. Here, we report that chemical resetting induces the expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co-expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions and to generate models of placental disorders.
Subject(s)
Pluripotent Stem Cells , Trophoblasts , Humans , Female , Pregnancy , Trophoblasts/metabolism , Transcriptional Activation , Placenta , Cell DifferentiationABSTRACT
Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm , Energy Metabolism/drug effects , Extracellular Matrix/drug effects , Lung Neoplasms/drug therapy , Mechanotransduction, Cellular/drug effects , Mitochondria/drug effects , Mitochondrial Dynamics/drug effects , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Transformed , Cell Line, Tumor , Cell-Matrix Junctions/drug effects , Cell-Matrix Junctions/metabolism , Cell-Matrix Junctions/pathology , Dynamins/metabolism , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mice, Inbred BALB C , Microfilament Proteins/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Nuclear Proteins/metabolism , Oxidation-Reduction , Oxidative Stress , Peptide Elongation Factors/metabolism , Tumor MicroenvironmentABSTRACT
Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions.
Subject(s)
Blastocyst/physiology , DNA Methylation/physiology , Embryonic Stem Cells/metabolism , STAT3 Transcription Factor/metabolism , Animals , Cell Differentiation , Cells, Cultured , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , Embryonic Stem Cells/physiology , Gene Expression Regulation , Histones/metabolism , Ketoglutaric Acids/metabolism , Leukemia Inhibitory Factor/metabolism , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Otx Transcription Factors/genetics , Otx Transcription Factors/metabolism , Pluripotent Stem Cells/metabolism , Promoter Regions, Genetic , STAT3 Transcription Factor/genetics , DNA Methyltransferase 3BABSTRACT
Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.
