NANOG-dependent function of TET1 and TET2 in establishment of pluripotency

Abstract

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.

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms

“…That study further showed that smallmolecule antagonists of RA signaling attenuate β-catenin and activate Wnt signaling in EpiSCs, thus providing a likely mechanism for how RA enhances reprogramming. We suggest that in addition to this effect, RA signaling enhances reprogramming by directly activating TET2 expression, a known reprogramming factor (16,18).…”

“…TET proteins can contribute to locus-specific demethylation in nESCs (1,14), and their depletion reduces the expression of pluripotency genes and increases methylation at their promoters (12,15). Furthermore, forced expression of TET1 and TET2 dramatically enhances iPSC reprogramming in a catalytically dependent manner (16)(17)(18). Nevertheless, the molecular signals that control TET activity in nESCs, and how they can be manipulated during reprogramming, are poorly characterized.…”

“TET-on” pluripotency

“…Co-expression of NANOG and TET1 increased the genome-wide levels of 5hmC. This process is dependent on NANOG-mediated recruitment of TET1 to NANOG-binding sites, suggesting that NANOG cooperates with TET1 in DNA demethylation at specific genomic loci during reprogramming [7]. Interestingly, another study discovered that TET2 and poly (ADP-ribose) polymerase-1 (PARP-1) cooperatively fine-tune epigenetic modifications and upregulate expression of the pluripotency genes Nanog and Esrrb in the early stage of somatic cell reprogramming [8], indicating a role of TET proteins in the regulation of pluripotency genes' expression as well as their activity.…”

“…However, little is known about its interacting partners that may contribute to the induction of pluripotency in pre-iPSCs. To identify the interacting partners of NANOG, Costa et al [7] utilized NANOG as bait to identify its physically associated partners via enhanced affinity purification followed by mass spectrometry. TET1 was identified as one of 27 high-confident partners in mESCs.…”

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles

“…For a long time the active process was unknown, however in 2011 studies showed that the responsibility for such process is the formation of 5-hydroxymethylcytosine (5-hmc), a cytosine variation, in which a -OH is added to the methylated cytosine by a protein from the Tet family; Tet 1, 2 or 3 (IQBAL et al, 2011;WOSSIDLO et al, 2011). Tet 1 and 2 have been related to embryonic stem cells pluripotency and depletion of Tet1 implicates in lineage differentiation difficulties (COSTA et al, 2013). Additionally, Tet 1 was detected in primordial germ cells highlighting its role in an undifferentiated profile (HAJKOVA et al, 2010), and Tet 3 is highly expressed in both oocyte and zygote, being responsible for 5-hmc increase on paternal pronucleus, while the maternal genome is protected by binding to PGC7 protein (also known as Dppa3 or Stella) (IQBAL et al, 2011;WOSSIDLO et al, 2011).…”