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Zfp281 functions as a transcriptional repressor for pluripotency of mouse embryonic stem cells.

ABSTRACT: Embryonic stem cells (ESCs) derived from preimplantation blastocysts have unique self-renewal and multilineage differentiation properties that are controlled by key components of a core regulatory network including Oct4, Sox2, and Nanog. Understanding molecular underpinnings of these properties requires identification and characterization of additional factors that act in conjunction with these key factors in ESCs. We have previously identified Zfp281, a Krüppel-like zinc finger transcription factor, as an interaction partner of Nanog. We now present detailed functional analyses of Zfp281 using a genetically ablated null allele in mouse ESCs. Our data show that while Zfp281 is dispensable for establishment and maintenance of ESCs, it is required for their proper differentiation in vitro. We performed microarray profiling in combination with previously published datasets of Zfp281 global target gene occupancy and found that Zfp281 mainly functions as a repressor to restrict expression of many stem cell pluripotency genes. In particular, we demonstrated that deletion of Zfp281 resulted in upregulation of Nanog at both the transcript and protein levels with concomitant compromised differentiation of ESCs during embryoid body culture. Chromatin immunoprecipitation experiments demonstrated that Zfp281 is required for Nanog binding to its own promoter, suggesting that Nanog-associated repressive complex(es) involving Zfp281 may fine-tune Nanog expression for pluripotency of ESCs.

SUBMITTER: Fidalgo M

PROVIDER: S-EPMC3272666 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Zfp281 functions as a transcriptional repressor for pluripotency of mouse embryonic stem cells.

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