scholarly journals TFEB regulates pluripotency transcriptional network in mouse embryonic stem cells independent of autophagy–lysosomal biogenesis

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Anderson Tan ◽  
Renuka Prasad ◽  
Eek-hoon Jho
Keyword(s):  
Target Genes ◽  
Embryonic Stem ◽  
Transcriptional Network ◽  
Double Knockout ◽  
Differentiation Markers ◽  
Lysosomal Activity ◽  
Stem Cell Maintenance ◽  
Protein Levels ◽  
Differentiated Cells ◽  
Pathogenic Factors

AbstractTranscription factor EB (TFEB), a well-known master regulator of autophagy and lysosomal biogenesis, is a member of the microphthalmia family of transcription factors (MiT family). Over the years, TFEB has been shown to have diverse roles in various physiological processes such as clearance for intracellular pathogenic factors and having developmental functions such as dendritic maturation, as well as osteoclast, and endoderm differentiation. However, in the present study, we propose a novel mechanism for TFEB governing pluripotency of mouse ESCs (mESCs) by regulating the pluripotency transcriptional network (PTN) in these cells. We observed high levels of TFEB mRNA and protein levels in undifferentiated mESCs. Interestingly, we found a reduction of Nanog and Sox2 levels in TFEB knockout (KO) mESCs while pluripotency was maintained as there was an upregulation of TFE3, a potent stem cell maintenance factor. In consistent, double knockout of TFEB/TFE3 (TFEB/3 DKO) reduced mESC pluripotency, as indicated by the loss of ESC morphology, reduction of ESC markers, and the emergence of differentiation markers. We further discovered that Nanog was a TFEB target gene in undifferentiated mESCs. TFEB also promoted sex-determining region Y-box2 (Sox2) transcription by forming a heterodimer with Sox2 in mESCs. Notably, Sox2, Oct4, and Nanog were also binding to the TFEB promoter and thus generating a feed-forward loop in relation to TFEB. Although high levels of nuclear TFEB are expected to enhance autophagy–lysosomal activity, undifferentiated mESC remarkably displayed low basal autophagy–lysosomal activity. Overexpression or knockout of TFEB did not affect the expression of TFEB lysosomal–autophagy target genes and TFEB also had a lesser binding affinity to its own lysosomal promoter-target genes in mESCs compared to differentiated cells. Collectively, these findings define a newly incorporative, moonlighting function for TFEB in regulating PTN, independent of its autophagy–lysosomal biogenesis roles.

Maintenance of Pluripotent Stem Cells is Influenced by Ser/Thr Metabolism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-43-SCI-43
Author(s):  
Lewis C. Cantley
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Isocitrate Dehydrogenase 1 ◽  
Advisory Committees ◽  
Metabolic Intermediates ◽  
Stem Cell Maintenance ◽  
Differentiated Cells ◽  
Methylation Of Dna

Abstract Recent studies have suggested not only that stem cells have different metabolic requirements than terminally differentiated cells, but also that metabolic intermediates may play a role in the maintenance of stem cells. It has long been clear that changes in acetylation and methylation of histones, as well as methylation of DNA play critical roles in deciding cell fate. The availability of critical intermediates in metabolism, especially S-adenosylmethionine (SAM), acetyl-CoA, nicotinamide adenine dinucleotide (NAD) and a-ketoglutarate play critical roles in modulating acetylation and methylation of histones and methylation of DNA. In the course of evaluating an unusual requirement of threonine (Thr) for the growth of murine embryonic stem cells, we found that metabolism of Thr to glycine (Gly) and the subsequent use of the methyl group of Gly for converting homocysteine to methionine is critical for maintaining high levels of SAM and low levels of S-adenosyl-homocysteine. Importantly, depletion of Thr from the media resulted in decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate. Demethylation of histones and DNA can also be controlled by metabolic intermediates. Mutated forms of isocitrate dehydrogenase 1 (IDH1) and IDH2 that drive acute myeloid leukemia (AML) and other cancers, produce an oncometabolite (2-hydrogyglutarate) that can compete with the a-ketoglutarate requirement for enzymes involved in hydroxy-methylation and subsequent demethylation of DNA and histones. Recent studies indicate that 2-hydroxyglutarate plays a role in blocking differentiation of cancer cells. These and other observations linking intermediates in metabolism to stem cell maintenance will be discussed. Disclosures Cantley: Agios Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

scholarly journals NRF2-dependent gene expression promotes ciliogenesis and Hedgehog signaling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ana Martin-Hurtado ◽  
Raquel Martin-Morales ◽  
Natalia Robledinos-Antón ◽  
Ruth Blanco ◽  
Ines Palacios-Blanco ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Embryonic Stem ◽  
Mtor Inhibitors ◽  
Gene Promoters ◽  
Protein Levels ◽  
Functional Connections ◽  
Differentiated Cells ◽  
Cilia Formation ◽  
And Function

Abstract The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implications.

scholarly journals Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor

2017 ◽  
Vol 114 (6) ◽  
pp. E922-E930 ◽  
Author(s):  
Cheng Wang ◽  
Stephen P. Goff
Keyword(s):  
Zinc Finger Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Degradation Pathway ◽  
Embryonic Cells ◽  
Polyubiquitin Chains ◽  
Protein Levels ◽  
Differentiated Cells ◽  
C Terminus ◽  
Differential Control

Replication of the murine leukemia viruses is strongly suppressed in mouse embryonic stem (ES) cells. Proviral DNAs are formed normally but are then silenced by a large complex bound to DNA by the ES cell-specific zinc-finger protein ZFP809. We show here that ZFP809 expression is not regulated by transcription but rather by protein turnover: ZFP809 protein is stable in embryonic cells but highly unstable in differentiated cells. The protein is heavily modified by the accumulation of polyubiquitin chains in differentiated cells and stabilized by the proteasome inhibitor MG132. A short sequence of amino acids at the C terminus of ZFP809, including a single lysine residue (K391), is required for the rapid turnover of the protein. The silencing cofactor TRIM28 was found to promote the degradation of ZFP809 in differentiated cells. These findings suggest that the stem cell state is established not only by an unusual transcriptional profile but also by unusual regulation of protein levels through the proteasomal degradation pathway.

scholarly journals Klf4 Cooperates with Oct3/4 and Sox2 To Activate the Lefty1 Core Promoter in Embryonic Stem Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7772-7782 ◽  
Author(s):  
Yuhki Nakatake ◽  
Nobutaka Fukui ◽  
Yuko Iwamatsu ◽  
Shinji Masui ◽  
Kadue Takahashi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Core Promoter ◽  
Es Cells ◽  
Embryonic Stem ◽  
Functional Screening ◽  
Transactivation Domain ◽  
Enhancer Element ◽  
Differentiated Cells ◽  
Promoter Dna

ABSTRACT Although the POU transcription factor Oct3/4 is pivotal in maintaining self renewal of embryonic stem (ES) cells, little is known of its molecular mechanisms. We previously reported that the N-terminal transactivation domain of Oct3/4 is required for activation of Lefty1 expression (H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki, Mol. Cell. Biol. 22:1526-1536, 2002). Here we test whether Lefty1 is a direct target of Oct3/4. We identified an ES cell-specific enhancer upstream of the Lefty1 promoter that contains binding sites for Oct3/4 and Sox2. Unlike other known Oct3/4-Sox2-dependent enhancers, however, this enhancer element could not be activated by Oct3/4 and Sox2 in differentiated cells. By functional screening of ES-specific transcription factors, we found that Krüppel-like factor 4 (Klf4) cooperates with Oct3/4 and Sox2 to activate Lefty1 expression, and that Klf4 acts as a mediating factor that specifically binds to the proximal element of the Lefty1 promoter. DNA microarray analysis revealed that a subset of putative Oct3/4 target genes may be regulated in the same manner. Our findings shed light on a novel function of Oct3/4 in ES cells.

scholarly journals Pluripotency of embryonic stem cells lacking clathrin-mediated endocytosis cannot be rescued by restoring cellular stiffness

2020 ◽  
Vol 295 (49) ◽  
pp. 16888-16896
Author(s):  
Ridim D. Mote ◽  
Jyoti Yadav ◽  
Surya Bansi Singh ◽  
Mahak Tiwari ◽  
Shinde Laxmikant V ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Physical Properties ◽  
Young’S Modulus ◽  
Actin Polymerization ◽  
Young's Modulus ◽  
Embryonic Stem ◽  
Actin Binding ◽  
Differentiation Markers ◽  
Differentiated Cells

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young's modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young's modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.

scholarly journals A Meta-Analysis Characterizing Stem-Like Gene Expression in the Suprachiasmatic Nucleus and Its Circadian Clock

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Dilshan Harshajith Beligala ◽  
Arpan De ◽  
Michael Eric Geusz
Keyword(s):  
Stem Cells ◽  
Circadian Clock ◽  
Suprachiasmatic Nucleus ◽  
Neural Circuit ◽  
Meta Analysis ◽  
Embryonic Stem ◽  
Stem Cell Maintenance ◽  
Differentiated Cells ◽  
Highly Expressed Genes ◽  
Phase Relationships

Cells expressing proteins characteristic of stem cells and progenitor cells are present in the suprachiasmatic nucleus (SCN) of the adult mammalian hypothalamus. Any relationship between this distinctive feature and the master circadian clock of the SCN is unclear. Considering the lack of obvious neurogenesis in the adult SCN relative to the hippocampus and other structures that provide neurons and glia, it is possible that the SCN has partially differentiated cells that can provide neural circuit plasticity rather than ongoing neurogenesis. To test this possibility, available databases and publications were explored to identify highly expressed genes in the mouse SCN that also have known or suspected roles in cell differentiation, maintenance of stem-like states, or cell-cell interactions found in adult and embryonic stem cells and cancer stem cells. The SCN was found to have numerous genes associated with stem cell maintenance and increased motility from which we selected 25 of the most relevant genes. Over ninety percent of these stem-like genes were expressed at higher levels in the SCN than in other brain areas. Further analysis of this gene set could provide a greater understanding of how adjustments in cell contacts alter period and phase relationships of circadian rhythms. Circadian timing and its role in cancer, sleep, and metabolic disorders are likely influenced by genes selected in this study.

scholarly journals Three classes of response elements for human PRC2 and MLL1/2-trithorax complexes

2017 ◽  
Author(s):  
Junqing Du ◽  
Brian Kirk ◽  
Jia Zeng ◽  
Jianpeng Ma ◽  
Qinghua Wang
Keyword(s):  
Target Genes ◽  
Cpg Islands ◽  
Embryonic Stem ◽  
Polycomb Group ◽  
Epigenetic Memory ◽  
Trithorax Group ◽  
Response Elements ◽  
Differentiated Cells ◽  
Dna Elements ◽  
First Time

SummaryPolycomb group (PcG) and trithorax group (TrxG) proteins are essential for maintaining epigenetic memory in both embryonic stem cells and differentiated cells. To date, how they are localized to hundreds of specific target genes within a vertebrate genome had remained elusive. Here, by focusing on short cis-acting DNA elements of single functions, we discovered, for the first time, to our knowledge, three classes of response elements in human genome: PcG response elements (PREs), MLL1/2-TrxG response elements (TREs) and PcG/TrxG response elements (P/TREs). We further demonstrated that, in contrast to their proposed roles in recruiting PcG proteins to PREs, YY1 and CpG islands are specifically enriched in TREs and P/TREs, but not in PREs. The three classes of response elements as unraveled in this study open new doors for a deeper understanding of PcG and TrxG mechanisms in vertebrates.

scholarly journals Hdac6 regulates Tip60-p400 function in stem cells

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Poshen B Chen ◽  
Jui-Hung Hung ◽  
Taylor L Hickman ◽  
Andrew H Coles ◽  
James F Carey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Stem Cells ◽  
Histone Deacetylase ◽  
Target Genes ◽  
Histone Acetyltransferase ◽  
Embryonic Stem ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Differentiated Cells

In embryonic stem cells (ESCs), the Tip60 histone acetyltransferase activates genes required for proliferation and silences genes that promote differentiation. Here we show that the class II histone deacetylase Hdac6 co-purifies with Tip60-p400 complex from ESCs. Hdac6 is necessary for regulation of most Tip60-p400 target genes, particularly those repressed by the complex. Unlike differentiated cells, where Hdac6 is mainly cytoplasmic, Hdac6 is largely nuclear in ESCs, neural stem cells (NSCs), and some cancer cell lines, and interacts with Tip60-p400 in each. Hdac6 localizes to promoters bound by Tip60-p400 in ESCs, binding downstream of transcription start sites. Surprisingly, Hdac6 does not appear to deacetylate histones, but rather is required for Tip60-p400 binding to many of its target genes. Finally, we find that, like canonical subunits of Tip60-p400, Hdac6 is necessary for robust ESC differentiation. These data suggest that Hdac6 plays a major role in the modulation of Tip60-p400 function in stem cells.

scholarly journals MicroRNA bta-miR-365-3p inhibits proliferation but promotes differentiation of primary bovine myoblasts by targeting the activin A receptor type I

2020 ◽  
Author(s):  
Dan Hao ◽  
Xiaogang Wang ◽  
Xiao Wang ◽  
Bo Thomsen ◽  
Xianyong Lan ◽  
...  
Keyword(s):  
Muscle Development ◽  
Target Genes ◽  
Heart Tissue ◽  
Nuclear Antigen ◽  
Type I ◽  
Differentiation Markers ◽  
Protein Coding ◽  
Protein Levels ◽  
Mrna Targets ◽  
And Function

Abstract Background MicroRNAs act as post-transcriptional regulators that repress translation or degrades mRNA transcripts. Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically bta-miR-365-3p, in muscle biology, and we studied its function to primary bovine myoblast proliferation and differentiation. Results We first show that bta-miR-365-3p is predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting showed that overexpression of bta-miR-365-3p significantly reduced the levels of cyclinD1 (CCND1), cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) but stimulated the expression of muscle differentiation markers MYOD1, MYOG. Moreover, downregulation of bta-miR-365-3p increased expression of CCND1, CDK2 and PCNA but decreased expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining showed that increased levels of bta-miR-365-3p suppressed cell proliferation but promoted myotube formation, whereas a decreased level of bta-miR-365-3p had opposite consequences. Finally, we determined that ACVR1 is a direct target of bta-miR-365-3p. Thus, dual luciferase gene reporter assays demonstrated that bta-miR-365-3p can bind to the 3'UTR of ACVR1 to regulate its expression. Consistently, knock-down of ACVR1 was associated with reduced CDK2, CCND1 and PCNA expression but increased MYOD1 expression. Conclusion Collectively these data suggest that bta-miR-365-3p represses proliferation but promotes differentiation of bovine myoblasts through a mechanism involving downregulation of ACVR1.

scholarly journals The ETS Transcription Factor GABPα Is Essential for Early Embryogenesis

2004 ◽  
Vol 24 (13) ◽  
pp. 5844-5849 ◽  
Author(s):  
Sika Ristevski ◽  
Debra A. O'Leary ◽  
Anders P. Thornell ◽  
Michael J. Owen ◽  
Ismail Kola ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Complex Function ◽  
Embryonic Stem ◽  
Transactivation Domain ◽  
Cellular Functions ◽  
Protein Levels ◽  
Ets Transcription Factor

ABSTRACT The ETS transcription factor complex GABP consists of the GABPα protein, containing an ETS DNA binding domain, and an unrelated GABPβ protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpα gene. Embryos homozygous for the null Gabpα allele die prior to implantation, consistent with the broad expression of Gabpα throughout embryogenesis and in embryonic stem cells. Gabpα+/− mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpα protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpα function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.

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