An expanded Oct4 interaction network: implications for stem cell biology, development, and disease

Abstract

The transcription factor Oct4 is key in embryonic stem cell identity and reprogramming. Insight into its partners should illuminate how the pluripotent state is established and regulated. Here, we identify a considerably expanded set of Oct4-binding proteins in mouse embryonic stem cells. We find that Oct4 associates with a varied set of proteins including regulators of gene expression and modulators of Oct4 function. Half of its partners are transcriptionally regulated by Oct4 itself or other stem cell transcription factors, whereas one-third display a significant change in expression upon cell differentiation. The majority of Oct4-associated proteins studied to date show an early lethal phenotype when mutated. A fraction of the human orthologs is associated with inherited developmental disorders or causative of cancer. The Oct4 interactome provides a resource for dissecting mechanisms of Oct4 function, enlightening the basis of pluripotency and development, and identifying potential additional reprogramming factors.

Figure 1

Analysis of Oct4-Interacting Proteins (A) Typical Oct4-FTAP and control purifications. Molecular weight markers (kDa) are shown. (B) Western blots confirming some of the interacting proteins identified by mass spectrometry. C, Western blot showing co-immunoprecipitation of endogenous Parp1 with Oct4 in the presence (B+) or absence (B-) of benzonase. In denotes whole cell extract. D, Workflow tracing the systems analyses of the Oct4 interactome. E, Network of protein-protein interactions within the Oct4 dataset. Blue circles are proteins downregulated upon ES cell differentiation. Red fill indicates proteins whose absence results in embryonic lethality in the mouse. See also Figure S1.

Figure 2

Expression of Oct4-associated proteins in ESCs and Differentiated Cell Types Based on Microarray Data Columns correspond to experimental samples, arranged as follows: embryonal carcinoma P19 (EC), ES cells (ES), differentiating embryonic stem cells (DES), adult stem cells (AS), differentiated adult stem cells (DAS), mixed cells (M), and differentiated cells (D). Average-linkage hierarchical clustering was performed to arrive at the final layout. Genes whose expression is significantly up or downregulated in differentiation are marked.

Figure 3

Transcriptional Regulation of Oct4-Associated Proteins (A) Scheme of transcription factor feedback regulation. (B) Regulatory network of targets of ESC transcription factors among Oct4 partners. Stem cell transcription factors and their target genes among Oct4-associated proteins are shown in dark and light gray, respectively. Oct4 and its regulatory targets are highlighted in blue. See also Figure S3.

Figure 4

Distribution of Phenotypes Caused by Mutations in the Genes Encoding Oct4-Associated Proteins Numbers are percentage of genes studied. Full data are shown in Table S5.

Figure 5

Schematic Model of the Oct4 Interactome