Interactome Profiling (RIME)

With RIME, mass spectrometry is used, along with immunoprecipitation of a target protein, to detect endogenous-interacting proteins in formaldehyde cross-linked cells.

Advantages of cell cross-linking include:

1. Preserves bona fide interactions allowing for more stringent washes and minimizing the detection of non-specific interactions.
2. Captures low-affinity interactions that are typically lost during washing.
3. Captures adjacent DNA binding proteins that participate in gene regulation but do not function through direct interaction with the targeted protein.

Benefits of RIME include:

1. Identify transcriptional co-factors/co-regulators.
2. Identify proteins complexed with epigenetic modifiers.
3. Detect low-affinity interactions.
4. Map protein interaction networks.
5. Validate identified proteins by ChIP-Seq and map global co-occupancy with the RIME target protein.
6. Understand how co-factors participate in differential gene regulation.
7. Identify the prominent co-occurrence of transcription factor binding at adjacent sites.

Figure 1: Venn Diagram of RIME treated MCF7 cells

RIME was performed to identify proteins that interact with the RNA polymerase machinery. Immunoprecipitations were performed with an antibody against POLR2A and two different amounts of cross-linked chromatin from MCF7 cells. Immunoprecipitated proteins were measured by mass spectrometry. The Venn diagram shows strong overlap of the detected proteins even in experiments using a 10 fold difference in starting material.

Advanced Application: RIME and ChIP-Seq

RIME was performed using BRD4 as the target protein and from the list of interacting proteins. DNA Topoisomerase 2A was identified as an interactor, which is supported by literature demonstrating that ligand-dependent Topoisomerases have been shown to be recruited to enhancers^†. Therefore, ChIP-Seq targeting BRD4 and TOP2A was performed to show co-localization. Expanding on RIME data with ChIP-Seq can help elucidate gene subsets that require different transcription factors or confirm co-localization of RIME-identified co-factors to the same genes.

Figures 1 & 2: ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.

ChIP-Seq was performed to map binding sites for the RIME-identified BRD4 interacting protein, TOP2A. TOP2A and BRD4 binding sites are shown and confirm co-localization at many sites across the genome. The top panel shows sites on chromosome 1 and the bottom panel shows sites on chromosome 3.

References

• ^†Puc, J. et al. Ligand-Dependent Enhancer Activation Regulated by Topoisomerase-I Activity, Cell. 2015 Oct 8;163(2):520-2.

Search our database of customer publications that have used our RIME services.