Abstract
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.
Highlights
Stem cells are often characterized by an intrinsic and flexible cellular potential to self-renew and differentiate into various functional cell types
Differentiation In western blot and real-time polymerase chain reaction (PCR) analyses, the levels of endogenous transcription factor EB (TFEB) mRNA and protein were observed to be high in undifferentiated cells, and the levels were subsequently reduced during differentiation induced by leukemia inhibitory factor (LIF) withdrawal (Supplementary Fig. 1a, b) or during embryonic body (EB) formation (Supplementary Fig. 1c, d)
The relative expression of MITF was low and TFEC was negligible in undifferentiated mouse ESCs (mESCs), which suggests that TFEB and transcription factor E3 (TFE3) are the major MiT family members in the undifferentiated mESCs
Summary
Stem cells are often characterized by an intrinsic and flexible cellular potential to self-renew and differentiate into various functional cell types. It is generally accepted that the self-renewal and differentiation potential of stem cells requires precise control of protein turnover and lysosome-mediated degradation of organelles[1]. Tan et al Cell Death and Disease (2021)12:343 factors (MiT family), is salient in controlling autophagylysosomal biogenesis and metabolics[12,13]. TFEB depletion has been reported to be associated with defective differentiation into osteoclast and endodermal lineages[15,16]. It was reported that the transcriptional activity of transcription factor E3 (TFE3), which is the most related to TFEB in the MiT family, is imperative for the maintenance of pluripotency by blocking pluripotency exit[17,18]. A potential role for TFEB in maintaining pluripotency of mESC has not been described
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