An Expanded Oct4 Interaction Network: Implications for Stem Cell Biology, Development, and Disease

Summary 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.

sequence of Pou5f1 within BAC RPCI23-213M12 by recombineering of a FTAP tag and subsequent integration of the modified BAC insert into the hprt tm(rmce1)Brd locus are schematically depicted. X: XhoI, N: NdeI, R: EcoRI, B: BamHI, Neo/Kan R : neomycin and kanamycin resistance gene, TK; thymidine kinase gene. The sensitivity (S-superscript) and resistance (R-superscript) of ES cell clones to the drugs puromycin (Puro), neomycin (Neo) and 6-thioguanine (6-TG) are indicated in the flow-diagram on the left. C, Western blots showing Oct4 expression (tagged and endogenous), and expression of markers of ES cells (Utf1, Nac1 and endogenous Oct4 itself). Gapdh was used as a loading control. The average ratio of endogenous Oct4 signal to tagged Oct4 (expressed from both alleles) is 3.032 (n=4, S.D.=0.39), as determined by pixel intensity analysis (ImageJ, NIH).  Table 1. Analysis of GO term enrichment of Oct4-associated proteins. In the GO Cellular Compartment category only terms with Bonferroni-corrected p < E-03 are shown for simplicity.
Full data is shown in Table S2.   Table 2. Sequence identity of mouse-human ortholog pairs. Box plot with Tukey whiskers, showing sequence identity percentages between the identified Oct4-associated proteins and their human orthologs, as determined by global sequence alignment. The difference is significant at p < 0.0001.
Genome-wide sequence identities for all mouse-to-human ortholog pairs contained in ENSEMBL are shown for comparison. Tables   Table S1, related to Table 1. Precursor ion mass accuracy. Mass accuracy of precursor ions of all peptides solely identifying a protein without additional support ("one-hit-wonders").

Generation of c-terminal FTAP tag recombineering constructs
The recombineering vector PL450 (Liu et al., 2003) was modified at the BstBI site by insertion of a SrfI restriction site as a double stranded oligonucleotide linker (pCOM). An RsrII fragment from the vector pKO SelectTK (Lexicon Genetics) was made blunt-ended by filling-in the overhang with Klenow and ligated into the modified PL450 at the SrfI site (pCOI1). The polylinker of the recombineering vector (pCOI1) was modified by cloning of a double stranded oligo nucleotide linker (BamHI overhang/ XhoI/ BamHI/ SacII) to generate a unique XhoI site.
The FTAP epitope tag (3xFLAG-2xTEV-CBP) sequence was synthesized as two DNA fragments by annealing two overlapping complementary oligonucleotide molecules using PCR with HiFi Supermix The filled-in double-stranded products were double restriction digested with BamHI/SacI for the 5' FTAP tag fragment and SacI/XhoI for the 3' FTAP tag fragment. The two fragments were cloned into the BamHI/XhoI digested recombineering vector (pCOI1) as a three way ligation to create pCTR9 ( Figure S1B).
The correctness of the FTAP tag within pCTR9 was confirmed by sequencing.

Generation of Pou5f1 recombineering construct
Homology arms for recombineering were PCR amplified from the Oct4 containing C57Black/6J derived BAC clone (RPCI 23-213M12) using the following oligonucleotides primers (restriction sites for generating sticky ends by digestion for ligation are indicated by underlining; NCBI m37 assembly chromosomal locations of the oligonucleotide sequence are indicated in brackets): The PCR products for the 5' and 3' homology were double-restriction digested with HindIII/BamHI and EcoRI/NdeI respectively and were sequentially cloned into the corresponding restriction sites of the recombineering vector to create pCTS1 ( Figure S1). The 5' homology arm creates an in-frame fusion between the Oct4 C-terminal coding sequence and the FTAP tag coding sequence, whilst deleting the stop codon.

Recombineering reaction
E. coli DH10B containing BAC clone RPCI 23-213M12 were made competent for recombineering by electroporation the miniλ prophage and selecting overnight at 32 o C on LB-agar plates with tetracycline (12.5μg/ml) and chloramphenicol (20μg./ml) (Court et al., 2003). A fragment for recombineering the FTAP tag sequence into the Oct4-containing BAC (RPCI 23-213M12) was generated by digesting clone pCTS1 with HindIII/NdeI. Correct recombination was confirmed by Southern analysis of BAC DNA using homology arm-specific DNA probes for all 6 tagged BAC clones tested.

Integration of modified BAC clones into the Hprt locus of ES cells by recombinase-mediated cassette exchange (RMCE)
ES cell cultures, electroporation and mini-Southern-blot analysis of ES cell clones were as described (Ramirez-Solis et al., 1993). The process for integrating single copy BAC transgenes at the Hprt locus by RMCE has been described previously (Prosser et al., 2008). For RMCE integration of tagged Oct4 BAC insert into hprt tm(rmce1)Brd allele of CCI18#1.6G, cells were co-transfected by electroporation (Biorad; 500 μF, 230 V) with pCAGGS-Cre (Araki et al., 1997) and the RPCI 23-213M12 BAC clone carrying an integrated copy of the FTAP tag cassette and neomycin resistance gene. Double resistant colonies were isolated after selection with G418 (200 μg/ml) for 5-6 d, and subsequent selection with 6-TG (10 μM) for 3-