Oct4 links multiple epigenetic pathways to the pluripotency network

Abstract

Oct4 is a well-known transcription factor that plays fundamental roles in stem cell self-renewal, pluripotency, and somatic cell reprogramming. However, limited information is available on Oct4-associated protein complexes and their intrinsic protein-protein interactions that dictate Oct4's critical regulatory activities. Here we employed an improved affinity purification approach combined with mass spectrometry to purify Oct4 protein complexes in mouse embryonic stem cells (mESCs), and discovered many novel Oct4 partners important for self-renewal and pluripotency of mESCs. Notably, we found that Oct4 is associated with multiple chromatin-modifying complexes with documented as well as newly proved functional significance in stem cell maintenance and somatic cell reprogramming. Our study establishes a solid biochemical basis for genetic and epigenetic regulation of stem cell pluripotency and provides a framework for exploring alternative factor-based reprogramming strategies.

Figure 1

Establishment of in vivo biotinylation system for affinity purification of Oct4 protein complexes in modified ZHBTc4 (ZO4B) ESCs. (A) Schematic depiction of the modified ZHBTc4 ESCs expressing biotinylated Oct4 protein. (B) Western blot analysis of relative expression levels of dox suppressible (^doxOct4) and constitutively active biotinylated Oct4 (^bioOct4) in the presence (+) or absence (−) of dox treatment. Top, anti-Oct4 blot; Bottom, SA-HRP blot. (C) Morphology of ZO4B4 ESCs in the presence (top) or absence (bottom) of dox. Images of colonies that are unstained (left panels) or stained for AP activity (right panels) are shown. (D) Normal expression levels of Oct4, Nanog, and Sox2 in ZO4B4, ZHBTc4, and J1 ESCs in the presence (+) or absence (−) of dox treatment. (E) Size exclusion chromatography (gel filtration) of nuclear extracts of J1 (top) and ZO4B4 (bottom) ESCs. Endogenous Oct4 protein complexes (^endOct4) in wild-type J1 ESCs were detected by anti-Oct4 western blot (top), and the Oct4 protein complexes in ZO4B4 ESCs (^bioOct4) were also detected by anti-Oct4 western blot (bottom).

Figure 2

Summary of Oct4-interacting proteins in mouse ESCs. (A) List of the 155 novel Oct4-associated proteins from affinity purification in ZO4B4 ESCs. (B) Comparison of our Oct4 interactome with the two published Oct4 network studies ^, . The overlapping proteins between our study and the two published studies are listed as blue, purple and red frames.

Figure 3

Connection of Oct4 with multiple epigenetic regulatory pathways. Summary of major protein complexes (red dotted circles) associated with the Oct4 interactome. Big filled circles with color are Oct4-interacting proteins that are also the RNAi hits from published genome-wide RNAi studies (see also Figure 5). Small filled circles with color are Oct4-interacting proteins identified in this study. Other components of the complexes not present in the Oct4 interactome are shown in gray small circles.

Figure 4

Validation of physical association of Oct4 with multiple epigenetic regulators. (A) Size exclusion chromatography analysis of Oct4-associated protein complexes. Nuclear extracts were fractionated in a DuoFlow BioLogic System and eluates were analyzed by western blotting with indicated antibodies to evaluate co-fractionation of endogenous ^endOct4 and biotinylated ^bioOct4 with PRC1, FACT, MutSalpha, MLL5-L, LSD1, Chromatin-remodeling complexes, as well as Arid3b. Migration of molecular markers is indicated above the panels, antibodies for WB are shown on the left and the complex name on the right. The two co-fractionation patterns of Oct4 with other factors are indicated with red and blue rectangles. (B) Validation of physical interactions between Oct4 and factors associated with multiple protein complexes. For validation of Oct4 interaction with Zmym2, Msh2, Ash2l, and Kif11, nuclear extracts from ZHBTc4 (control) or ZO4B4 (^bioOct4) cells were incubated with SA-beads as described in Materials and Methods. Precipitated samples were analyzed by western blotting with indicated antibodies. For IP with anti-Oct4 antibody, nuclear extracts were prepared from J1 ESCs (for Supt16h, Msh6, Ppp1cc, Arid3b) or ^bioArid3b ESCs, and endogenous Oct4 complexes were immunoprecipitated with an Oct4 antibody followed by western blot with indicated antibodies or SA-HRP. The interaction of Ring1B with Oct4 was validated by SA-IP of nuclear extracts from ^bioRing1B, and control BirA ESCs followed by western blot with anti-Oct4.

Figure 5

Functional validation of the Oct4-interacting proteins by RNAi. (A) The Oct4 interactome is enriched for positive RNAi hits required for stem cell maintenance. Green and red circles denote RNAi studies in mESCs ^{, ,} and hESCs , respectively. Blue circles denote RNAi studies in both mESCs and hESCs. The P value is ∼2 × 10⁻⁹ from Fisher exact test. (B) The list of Oct4-interacting proteins whose encoding genes are also the positive RNAi hits shown in A.

Figure 6

Enrichment of the Oct4 interactome for factors whose encoding genes are downstream targets of the core pluripotency factors Nanog, Oct4, and Sox2. We utilized previously published genome-wide binding data for Sox2, Oct4, and Nanog in mESCs ^, to generate the protein-DNA regulatory network. Target genes are represented as smooth-edged rectangles and upstream transcription factors are represented as filled ovals. Arrows emanating from each upstream transcription factor refer to its binding to the indicated genes. The total number of genes occupied by each of the core factors is indicated.