Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein Nanog1–2, which plays an essential role in establishing ground state pluripotency during somatic cell reprogramming3–4. While the genomic occupancy of Nanog has been extensively investigated, comparatively little is known about Nanog-associated proteins5 and their contribution to the Nanog-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identified 27 high-confidence protein interaction partners of Nanog in mouse ES cells. These consist of 19 novel partners of Nanog that have not been reported before including the Ten eleven translocation (Tet) family methylcytosine hydroxylase Tet1. We confirmed physical association of Nanog with Tet1, and demonstrated that Tet1, in synergy with Nanog, enhances the efficiency of reprogramming. We also found physical association and reprogramming synergy of Tet2 with Nanog, and demonstrated that knockdown of Tet2 abolishes the reprogramming synergy of Nanog with a catalytically deficient mutant of Tet1 (Tet1Mut). These results indicate that the physical interaction between Nanog and Tet1/2 proteins facilitates reprogramming in a manner that is dependent on Tet1/2's catalytic activity. Tet1 and Nanog co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in ES cells, and Tet1 binding is reduced upon Nanog depletion. Co-expression of Nanog and Tet1 results in expression priming of and increased 5hmC levels at top ranked common targets Esrrb and Oct4 before reprogramming to naïve pluripotency. We propose that Tet1 is recruited by Nanog to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of Nanog and uncover a novel role for 5mC hydroxylases in the establishment of naïve pluripotency.
SUMMARY Epigenetic and epitranscriptomic networks have important functions in maintaining pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However the mechanisms integrating the actions of these distinct networks are only partially understood. Here, we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates transcription of key pluripotency genes, and modulates N6-methyladenosine (m6A) deposition on their transcripts by sequestering the enzyme m6A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m6A RNA levels, and enhances m6A modification of Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3-associated, and is enriched at promoters of m6A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m6A RNA modification to insure ESC identity.
In mammals, all somatic development originates from lineage segregation in early embryos. However, the dynamics of transcriptomes and epigenomes acting in concert with initial cell fate commitment remains poorly characterized. Here we report a comprehensive investigation of transcriptomes and base-resolution methylomes for early lineages in peri- and postimplantation mouse embryos. We found allele-specific and lineage-specific de novo methylation at CG and CH sites that led to differential methylation between embryonic and extraembryonic lineages at promoters of lineage regulators, gene bodies, and DNA-methylation valleys. By using Hi-C experiments to define chromatin architecture across the same developmental period, we demonstrated that both global demethylation and remethylation in early development correlate with chromatin compartments. Dynamic local methylation was evident during gastrulation, which enabled the identification of putative regulatory elements. Finally, we found that de novo methylation patterning does not strictly require implantation. These data reveal dynamic transcriptomes, DNA methylomes, and 3D chromatin landscapes during the earliest stages of mammalian lineage specification.
A number of key regulators of mouse embryonic stem (ES) cell identity, including the transcription factor Nanog, show strong expression fluctuations at the single cell level. The molecular basis for these fluctuations is unknown. Here we used a genetic complementation strategy to investigate expression changes during transient periods of Nanog downregulation. Employing an integrated approach, that includes high-throughput single cell transcriptional profiling and mathematical modelling, we found that early molecular changes subsequent to Nanog loss are stochastic and reversible. However, analysis also revealed that Nanog loss severely compromises the self-sustaining feedback structure of the ES cell regulatory network. Consequently, these nascent changes soon become consolidated to committed fate decisions in the prolonged absence of Nanog. Consistent with this, we found that exogenous regulation of Nanog-dependent feedback control mechanisms produced more a homogeneous ES cell population. Taken together our results indicate that Nanog-dependent feedback loops have a role in controlling both ES cell fate decisions and population variability.
Ten-eleven translocation (TET) proteins play key roles in regulating the methylation status of DNA through oxidizing methylcytosines (5mC), generating 5-hydroxymethylcytosines (5hmC) that can both serve as stable epigenetic marks and participate in active demethylation. Unlike the other TET-family members, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We find that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and posttranscriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.
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