Pluripotency, the ability of a cell to give rise to all cell types of an organism, is a fundamental characteristic of embryonic stem cells (ESCs). The basis of pluripotency resides in conserved transcriptional regulatory networks 1, 2
and protein interaction networks 3, 4, 5
of numerous transcription factors (TFs) and epigenetic regulators, which act together to repress developmental genes and activate stemness genes in ESCs. Oct4, Sox2, and Nanog are well-known key components of the core regulatory network that governs ESC pluripotency, and epigenetic regulators such as the polycomb group proteins, SWI/SNF proteins, and Mi-2/NuRD complex proteins also play important roles in maintaining pluripotency 3
. Understanding the interactions among these pluripotency TFs and epigenetic cofactors is critical for maintaining as well as directly differentiating pluripotent stem cells.
Efforts to decipher the molecular basis for pluripotency of ESCs have culminated in the discovery of a set of reprogramming factors that, when ectopically expressed, directly convert somatic cells to so-called 'induced pluripotent stem cells' (iPSCs) 6
. These reprogramming factors, namely Oct4 and Sox2 in combination with Klf4 and c-Myc 6
, or Nanog and Lin28 7
, are also known to be important factors for self-renewal and pluripotency of ESCs. Nanog can promote transfer of pluripotency after cell fusion 8
and ensure direct reprogramming of somatic cells to the pluripotent ground state 7, 9
. Various combinations and replacement of reprogramming factors with other pluripotency factors or small molecules have been achieved in reprogramming different types of somatic cells. However, the reprogramming process is slow and inefficient, and improved reprogramming requires additional epigenetic modifiers, suggesting the existence of epigenetic barriers to somatic cell reprogramming. Oct4 has largely remained an irreplaceable factor with only one exception 10
. In that exceptional case, the orphan nuclear receptor Nr5a2 (also known as Lrh-1), a known Oct4 activator, replaces Oct4 in the derivation of iPSCs from mouse somatic cells and enhances reprogramming efficiency partly through Nanog activation 10
, consistent with a fundamental role of Nanog and Oct4 in stem cell pluripotency and somatic cell reprogramming. An improved understanding of genetic and epigenetic mechanisms by which the core ESC factors, Nanog and Oct4 in particular, regulate pluripotency should help in designing alternative or improved reprogramming strategies and providing mechanistic insights into somatic cell reprogramming.
Genetic studies 11, 12, 13
have defined the homeodomain transcription factor Nanog as the key self-renewal regulator that is essential for early development and for safeguarding the ground-state pluripotency of ESCs. ESCs lacking Nanog exhibit compromised self-renewal and tend to differentiate toward the primitive endoderm lineage 12
. In contrast, enforced expression of Nanog results in enhanced self-renewal at the expense of differentiation propensity 14
. To understand how Nanog functions in regulating self-renewal and maintaining/promoting pluripotency, we have tested and established an in vivo
biotinylation strategy for affinity purification of protein complexes associated with Nanog, and constructed the first protein interaction network in mouse (m) ESCs (the Nanog interactome) 3
. The Nanog interactome encompasses Oct4 and multiple genetic and epigenetic regulators that individually and combinatorially contribute to stem cell pluripotency 3
. Recent reports suggest that Oct4 is essential for integrating the epigenetic machinery into the pluripotency network. For example, Oct4 cooperates with Nanog and Sox2 to repress Xist
(X-inactive specific transcript) and thus couples X inactivation reprogramming to pluripotency 15
. Oct4 also interacts with several polycomb group proteins (e.g., Ring1B, Rybp) as part of the Nanog interactome 3
to maintain pluripotency 16
. In addition, Oct4 controls the chromatin architecture of ESCs by directly regulating downstream target genes encoding the H3K9 demethylases Jmjd1a and Jmjd2c, which modulate the H3K9 methylation status of the pluripotency factors Tcl1 and Nanog, respectively, to maintain stem cell identity 17
. Limited studies have been performed to dissect the biochemical basis for Oct4's diverse roles in both genetic and epigenetic regulation of stem cell pluripotency. The most notable ones are two recently published biochemical studies that used FLAG-based affinity purification of Oct4 complexes in mouse embryonic stem cells (mESCs) 4, 5
. These studies resulted in discouragingly few overlapping Oct4 partners 18
, and left an open question of whether we have identified the bona fide
Oct4 interactome in ESCs.
Here we report an extended Oct4 interactome composed of a much larger repertoire of interacting proteins than previously reported 3, 4, 5
using an advanced affinity purification approach with demonstrated effectiveness for affinity purification of protein complexes in ESCs 3, 19, 20
. We discovered and confirmed physical association and functional significance of a number of novel Oct4 partners. Our study provides solid biochemical evidence and strong functional validation that Oct4 is critical for epigenetic regulation of stem cell pluripotency. We demonstrate that the Oct4 interactome is connected with multiple chromatin remodeling and epigenetic regulatory protein complexes that are important for stem cell maintenance, pluripotency, and somatic cell reprogramming (iPSC generation).