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ChIP-SICAP Reveals Pluripotency’s Secrets

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Review of  “Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins” from Molecular Cell by Stuart P. Atkinson

Identifying factors which bind to specific DNA elements has helped to construct a vast and intricate network of interacting factors which control the pluripotent state of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The pluripotency-associated transcription factors Oct4, Sox2, and Nanog (OSN) represent some of the most important DNA binding elements; but what other factors help them to carry out their function?

To answer this question, researchers from the laboratory of Jeroen Krijgsveld (EMBL, Heidelberg, Germany) have devised a new chromatin immunoprecipitation (ChIP)-based assay to identify factors which colocalize with OSN to control gene regulation. Rafiee et al now report in a new Molecular Cell study on how the selective isolation of chromatin-associated proteins through ChIP (ChIP-SICAP) can reveal pluripotency’s secrets [1].

Briefly, the ChIP-SICAP assay targets endogenous proteins (such as OSN) with a specific antibody, the associated DNA is biotinylated, the chromatin and interacting proteins retrieved using streptavidin beads, and proteins identified  by mass spectrometry. The authors found that this strategy permitted the specific enrichment of chromatin-bound partners of a target protein without the presence of common contaminants, functioning better than other related strategies such as ChIP-Mass Spectrometry (ChIP-MS) [2, 3] and rapid immunoprecipitation-mass spectrometry of endogenous proteins (RIME) [4].

So can ChIP-SICAP reveals pluripotency’s secrets?  The authors first compared OSN-associated factors following ChIP-SICAP in mESCs grown under standard self-renewal culture conditions and  ground state (or “naïve”) pluripotency conditions. This revealed a pluripotent state-dependent association of transcription factors and epigenetic modifiers with OSN and also identified the Trim24 (tripartite motif-containing 24) protein as an important new pluripotency network member. Trim24 is a chromatin-binding factor with known roles in differentiation, development, and tissue homeostasis, but no known roles in pluripotency. However, in this study, the authors demonstrated that Trim24 aided OSN-mediated repression of developmental genes and activation of cell cycle genes via interactions with enhancer elements.

A great new technique and a valuable new piece of the pluripotency network revealed; how can ChIP-SICAP help your research? Trim24 represents just one of the 400 proteins found to interact with OSN and the comparisons between mESC pluripotent states will also surely lead to further exciting findings. The authors also note that their ChIP-SICAP will combine well with other cell types and a variety of transcription factors, transcriptional regulators, and post-translationally modified histones, and so could do much more than reveal pluripotency’s secrets.

References

  1. Rafiee MR, Girardot C, Sigismondo G, et al. Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins. Mol Cell 2016;64:624-635.
  2. Engelen E, Brandsma JH, Moen MJ, et al. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry. Nat Commun 2015;6:7155.
  3. Ji X, Dadon DB, Abraham BJ, et al. Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions. Proc Natl Acad Sci U S A 2015;112:3841-3846.
  4. Mohammed H, Taylor C, Brown GD, et al. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat Protoc 2016;11:316-326.