Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia and behavioral changes. Extracellular deposition of amyloid plaques (Aβ) and intracellular deposition of neurofibrillary tangles in neurons are the major pathogenicities of AD. However, drugs targeting these therapeutic targets are not effective. Therefore, novel targets for the treatment of AD urgently need to be identified. Expression of the mesoderm-specific transcript (Mest) is regulated by genomic imprinting, where only the paternal allele is active for transcription. We identified hypermethylation on the Mest promoter, which led to a reduction in Mest mRNA levels and activation of Wnt signaling in brain tissues of AD patients. Mest knockout (KO) using the CRIPSR/Cas9 system in mouse embryonic stem cells and P19 embryonic carcinoma cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing shMest or inducible KO system causes neurodegeneration. Notably, depletion of Mest in primary cortical neurons of rats leads to tau phosphorylation at the S199 and T231 sites. Overall, our data suggest that hypermethylation of the Mest promoter may cause or facilitate the progression of AD.
Transcription 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.
Background and Objectives Tcfs and Lef1 are DNA-binding transcriptional factors in the canonical Wnt signaling pathway. In the absence of β -catenin, Tcfs and Lef1 generally act as transcriptional repressors with co-repressor proteins such as Groucho, CtBP, and HIC-5. However, Tcfs and Lef1 turn into transcriptional activators during the interaction with β -catenin. Therefore, the activity of Tcfs and Lef1 is regulated by β -catenin. However, the intrinsic role of Tcfs and Lef1 has yet to be examined. The purpose of this study was to determine whether Tcfs and Lef1 play differential roles in the regulation of self-renewal and differentiation of mouse ES cells. Methods and Results Interestingly, the expression of Tcfs and Lef1 was dynamically altered under various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcf and Lef1 may vary in ES cells. Ectopic expression of Tcf1 or the dominant negative form of Lef1 (Lef1-DN) contributes to ES cells to self-renew in the absence of leukemia inhibitory factor (LIF), whereas ectopic expression of Tcf3, Lef1 or Tcf1-DN did not support ES cells to self-renew. Ectopic expression of either Lef1 or Lef1-DN blocked neuronal differentiation, suggesting that the transient induction of Lef1 was necessary for the initiation and progress of differentiation. ChIP analysis shows that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog. Conclusions The overall data suggest that Tcf1 plays a critical role in the maintenance of stemness whereas Lef1 is involved in the initiation of differentiation.
Background: The expression of mesoderm-specific transcript (Mest) is regulated by genomic imprinting where only the paternal allele is active for transcription. Mest is a candidate gene for Silver-Russell Syndrome, and hypermethylation of Mest promoter is associated with oligozoospermia. In addition, Loss of imprinting (LOI) is often associated with various neurodegenerative diseases and cancers. Since aberrant Mest hypermethylation and LOI are implicated in various diseases, it is vital to study Mest promoter hypermethylation and its functional role in AD patients. Methods: To assess the Mest promoter methylation, bisulfite sequencing technique was used. CRIPSR/Cas9 system was used to generate the Mest knockout (KO) in both embryonic carcinoma and mouse embryonic stem cells. Lentiviral Split-Cas9 system (LSC-5) was employed to generate inducible Mest KO in neurons. Results: We found that Mest promoter is hypermethylated, which led to the reduction of Mest mRNA levels and activation of Wnt signaling in the brain tissues of AD patients. Mest KO in both embryonic carcinoma and mouse embryonic stem cells leads to neuronal differentiation arrest. Depletion of Mest in primary hippocampal neurons via lentivirus expressing sh-Mest or inducible KO system caused neurodegeneration. Notably, depletion of Mest in rat primary cortical neurons leads to tau phosphorylation at S199 and T231 sites. Conclusion: Our study has unfolded the epigenetic modification; Mest promoter hypermethylation in AD. Biochemically, we have linked this with Wnt signaling activation and Tau phosphorylation in neurons.
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