Maple-bacon, chocolate, and hedgehog? The list of potato chip flavors keeps on getting longer and crazier. Also getting longer is the list of different ChIP assay varieties (that’s chromatin immunoprecipitation for the uninitiated), with the most interesting new “taste sensation” emanating from the lab of Jeroen Krijgsveld (DFKZ/EMBL, Heidelberg, Germany).
While chips are good for scooping salsa and accompanying a cold brew, the Krijgsveld’s new ChIP assay has a somewhat wider potential. In their new paper, Rafiee et al. describe the selective isolation of chromatin-associated proteins via ChIP (ChIP-SICAP) to identify factors that colocalize with a DNA binding factor of choice and regulate gene expression.
Follow these steps to get a flavor of what ChIP-SICAP is all about (Molecular Cell DOI: 10.1016/j.molcel.2016.09.019)
The ChIP-SICAP Assay
In brief, the ChIP-SICAP assay (as depicted above) isolates formaldehyde cross-linked protein-DNA complexes via an antibody hungry for a factor of choice. End labeling of DNA fragments from isolated complexes with biotinylated nucleotides permits capture by streptavidin beads and allows for stringent washing to remove all contaminants before mass spectroscopy identifies associated proteins. The assay also comes with a side dish of DNA for some high-quality ChIP-sequencing that complements the protein interaction data.
The authors employed their new ChIP flavor to study proteins associated with pluripotency-associated transcription factors (Oct4, Sox2, and Nanog [OSN]) in mouse embryonic stem cells (mESCs). Initial comparisons of cells grown under classical mESC culture conditions to cells grown in ground state (or “naïve”) pluripotency conditions revealed a pluripotent state-dependent association of transcription factors and epigenetic modifiers.
Additionally, this comparison also helped to identify the Trim24 (tripartite motif-containing 24) protein as an important new pluripotency network member. Trim24, a chromatin-binding factor with known roles in differentiation, development, and tissue homeostasis, had no previously recognized roles in the regulation of 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.
With a great new technique and a valuable new piece of the pluripotency network revealed, could ChIP-SICAP become your new favorite assay flavor? Trim24 represents just one of the 400 proteins found to interact with OSN and this, combined with the detailed comparisons between mESC pluripotent states, will surely lead to further exciting discoveries. The authors also state that their new ChIP flavor will combine well with other cell types and a variety of transcription factors, transcriptional regulators, and post-translationally modified histones.
For a flavorsome read, set your taste buds to “science” and head on over to Molecular Cell, October 2016.