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Lineage-specific enhancer insertions regulate Prdm14 to drive the rapid transition from naïve to formative pluripotency in rodents

ABSTRACT: The network of transcription factors is dynamically reorganized during the transition from naïve to formative pluripotency. In mice, Prdm14 is expressed in naïve pluripotent cells but rapidly downregulated upon exit from the naïve state. In contrast, PRDM14 expression persists throughout pluripotency transitions in non-rodent mammals, including pigs and humans. Here, we investigate the molecular mechanisms underlying the rodent-specific expression of Prdm14. Using CRISPR/Cas9-mediated deletions, we demonstrated that OCT4 and TFCP2L1 recognition sequences within Muroidea-specific cis-regulatory elements located downstream of Prdm14 are essential for its transcriptional upregulation required for the upregulation of Prdm14 in naive embryonic stem cells (ESCs). Loss of these enhancers suppresses Pramel7 induction and impairs degradation of UHRF1, thereby attenuating global DNA demethylation under 2iL conditions. Moreover, deletion of PRDM14-binding motifs in Muroidea-specific enhancers disrupts its negative feedback loop, resulting in delayed transition from the naïve to formative pluripotent state.

ORGANISM(S): Mus musculus

PROVIDER: GSE309484 | GEO | 2025/11/20

REPOSITORIES: GEO

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Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.

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Lineage-specific enhancer insertions regulate Prdm14 to drive the rapid transition from naïve to formative pluripotency in rodents

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