Successful co-immunoprecipitation of Oct4 and Nanog using cross-linking

Abstract

The transcription factors Oct4 and Nanog are essential for the maintenance of an undifferentiated and pluripotent state in early embryonic cells, embryonic stem cells and embryonal carcinoma cells in humans and mice. These factors are co-localized to promoters of more than 300 genes, and synergistically regulate their activities. Currently, the molecular interaction between these two factors has not been well-characterized. During attempts to co-immunoprecipitate Oct4 and Nanog we found that cross-linking with dithiobis[succinimidylpropionate] was necessary to maintain their interaction. This result was supported by gel filtration analysis. Surprisingly, formaldehyde, a cross-linker commonly used during chromatin immunoprecipitation of Oct4 and Nanog, did not preserve the complex. Our findings demonstrate the effectiveness of using DSP to mitigate the instability of the interaction between these two particular proteins. Additionally, this solution may potentially allow us to identify novel members of the Oct4-Nanog complex, leading to better understanding of the regulatory mechanisms behind pluripotency.

Combinatorial binding of transcription factors in the pluripotency control regions of the genome

“…The association between strong NANOG and POU5F1 binding is compelling and is supported by the finding that NANOG and POU5F1 can be coimmunoprecipitated in vivo (Zhang et al 2007). The optimal binding location of NANOG lies 3-4 nt apart from the octamer motif, raising the possibility that, at least on these substrates, NANOG may not directly contact POU5F1.…”

“…Precise POU5F1 levels are also critical to maintaining pluripotency and are at least partially controlled by SOX2, a factor that can heterodimerize to potentially change POU5F1 binding specificity (Niwa et al 2000;Remenyi et al 2003). POU5F1 can be regarded as a central pillar in that SOX2 and NANOG interact with POU5F1 but are not known to interact with each other; POU5F1 can be regarded as the hub of this pluripotency network (Zhang et al 2007).…”

“…For example, POU2F1 and POU2F2, but not other POU factors, interact with the DNA binding tissue-specific cofactor POU2AF1 (also known as OCA-B and OBF-1) (Gstaiger et al 1996). POU5F1 is known to dimerize with SOX2 and can be immunoprecipitated with NANOG (Zhang et al 2007). …”

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1

“…Bound proteins were separated on an SDS-8% polyacrylamide gel and analyzed by Western blotting. For immunoprecipitation experiments using cross-linked nuclear extract, 90% confluent plates of HeLa S3 cells were treated with 2 mM dithiobis(succinimidyl propionate) cross-linking agent (Pierce) for 2 h at 4°C before stopping the reaction with 20 mM Tris-HCl (pH 8.0) at 25°C for 20 min (25). Approximately 250 g of cross-linked HeLa nuclear extract was incubated with either preimmune or immune anti-PRMT7 antibody as described above, and bound proteins were washed seven times with 1 ml of washing buffer (40 mM TrisHCl (pH 8.0), 350 mM NaCl, 0.75% Nonidet P-40 and 0.05% SDS, 0.25 mM DTT, 0.5 mM PMSF, 1% aprotinin) before they were analyzed by Western blotting.…”

“…In contrast, association between endogenous PRMT7 and BRG1-based hSWI/ SNF complex was readily detectable using nuclear extracts from HeLa S3 cells treated with the cross-linking agent dithiobis(succinimidyl propionate), which can enhance interaction between neighboring proteins (Fig. 1D) (25). Both BRG1 and BAF155 were detected in immune but not preimmune anti-PRMT7 immunoprecipitates under high salt washing conditions (350 mM NaCl).…”