Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

With the completion of the human genome sequencing, decoding the functional elements is a major challenge. Computational approaches have the power to identify conserved DNA regulatory elements; however, computational strategies cannot confidently predict the proteins that bind to these elements. Identification of the interaction networks between the DNA functional elements and the human proteome requires extensive predictions and powerful high throughput techniques. Hu and colleagues undertook a large-scale analysis of protein-DNA interactions (PDIs) using a protein microarray composed of 4191 unique full length human proteins, encompassing 90% of the annotated transcriptions factors (TFs) and members of many other protein categories, such as RNA-binding proteins, chromatin-associated proteins, nucleotide-binding proteins, transcription co-regulators, mitochondrial proteins and protein kinases [18] . The protein microarrays were probed with 400 predicted and 60 known DNA motifs. As a result, a total of 17,718 PDIs were identified. Many known PDIs and a large number of new PDIs for both well characterized and predicted TFs were recovered, as well as new consensus sites for human TFs. Surprisingly, over 300 proteins that do not encode any known DNA-binding domains showed sequence-specific PDIs, suggesting that many human proteins may bind specific DNA sequences as a secondary function. To further investigate whether the DNA-binding activities of these unconventional DNA binding proteins (uDBPs) were physiologically relevant, Hu et al. carried out in-depth analysis on a well-studied protein kinase, Erk2, to determine the potential mechanism behind its DNA-binding activity [18] . Using a combination of in vitro and in vivo approaches, such as electrophoretic mobility shift assays (EMSA), luciferase assays, mutagenesis, and chromatin immunoprecipitation (chIP), they demonstrated that the DNA-binding activity of Erk2 is independent of its protein kinase activity and it acts as a transcription repressor of transcripts induced by interferon gamma signaling [18] . This approach allows for sophisticated network mapping of protein-DNA interactions and enables the discovery of the uncharacterized DNA-binding proteins. The emergence of uDBPs strengthens the ability to piece together the machinery involved in transcriptional regulation.