The role of Trim71 in cell fate determination of embryonic stem cells and germline development

Abstract

The formation of complex tissues and organs from a limited set of stem and progenitor cells requires precise regulation of the balance between stem cell maintenance and differentiation. In the present study we introduce Trim71 as a novel regulator of this balance. Trim71 heterozygous mice exhibit lifelong growth retardation, while complete deficiency of Trim71 causes defects in neural tube closure and embryonic lethality. To unravel the molecular processes regulated by Trim71, we derived mESC lines in which the Trim71 gene can be conditionally mutated <em>in vitro</em>. Interestingly, Trim71 knockout mESCs display no deficits in the maintenance of stemness in steady-state conditions. However, upon induction of differentiation a premature upregulation of neural marker gene expression can be observed, which is in accordance with the already described phenotype of the Trim71 knockout mice. High-throughput sequencing of mRNAs and miRNAs in Trim71-deficient and control mESCs revealed that neuroectodermal priming already occurred in undifferentiated mESCs. While genes associated with stemness regulation were majorly unaffected, Trim71 deficiency led to an upregulation of both mRNAs and miRNAs involved in neuroectodermal development. We could show that Trim71 reduces the stability of specific targetmRNAs via response elements located in their respective 3’UTRs. <br /> Furthermore, we found that Trim71 is involved in a feedback mechanism regulating the expression of the pro-differentiation miRNA let-7. We propose that Trim71 cooperates with the stem cell protein Lin28 to facilitate repression of let-7 miRNA maturation in undifferentiated mESCs. We observed co-localization and physical interaction of Trim71 with Lin28 and found functional cooperation of both proteins in different cell types which led to the repression of let-7 maturation. These results suggested that Lin28 was necessary and sufficient for the function of the Trim71 protein as a let-7 repressor, and this was confirmed by ablating the Lin28 gene in mESCs using TALEN-based genome engineering. Importantly, Trim71 and Lin28 are both relevant targets of let-7 and are downregulated once differentiation is induced. Therefore, our data establish a role of Trim71 as an important regulator of the bistable let-7/Lin28 switch in mESCs. <br /> Last, we were interested to investigate the role of Trim71 in the adult organism. For this purpose, we used the conditional Trim71 allele to generate a germ cell-specific Trim71 knockout mouse. Deletion of Trim71 in the germline leads to infertility of both male and female mice. Whereas initial germ cell fate determination seems not to be impaired in Trim71 knockout mice, we found that Trim71 is essential for the survival of these cells during embryonic development. Moreover, we showed that Trim71 expression is retained in the spermatogonial stem cell population of the testis in adult mice. <br /> Taken together, the acquired data reveal important aspects of Trim71 functionality that might further the understanding of cell fate programming and maintenance in both normal and pathophysiological conditions.