Mapping transcription factor interactome networks using HaloTag protein arrays

Abstract

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literaturecurated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.protein arrays | interactome | hormone | systems biology | Arabidopsis thalianaA major objective in the postgenomic era is to assign detailed molecular function(s) to the many protein-coding genes that remain uncharacterized even in model organisms such as the reference plant Arabidopsis thaliana (1). It is estimated that Arabidopsis contains more than 2,000 transcription factors and transcriptional regulators (hereafter "TFs"), most of which are uncharacterized (2). TFs function as key players in plant hormone signal transduction pathways and are responsible for directing the widespread changes in gene expression that are essential for the regulation of growth and development in plants (3, 4). The TFs within these transcriptional regulatory networks do not work independently but rather undergo complex interactions with other proteins (2). Understanding how these TFs interact with other proteins will ultimately lead to a greater comprehension of biological systems.For determination of physical protein-protein interactions (PPIs), protein microarrays (5-10) are complementary to other PPI technologies such as the yeast two-hybrid system (Y2H) (11) and protein complex purification coupled with mass spectrometry (AP-MS) (12, 13). With conventional protein microarrays it is necessary to purify thousands of in vivo expressed proteins and to spot these purified proteins on a solid surface (5-8). In contrast, in situ synthesis protein microarray technologies simplify protein microarray fabrication by circumventing the steps of in vivo protein expression and purification (9,10,14,15). This streamlining facilitates an increase in the number of target genes that can be assayed, allowing for thousands of protein-encoding plasmids to be spotted at lower cost and in less time. Such ...

Mapping genome-wide transcription-factor binding sites using DAP-seq

“…Both protein expression and subsequent binding to the beads (verified by Western blot; Box 3) are crucial for a successful experiment. For high-throughput processing of samples, we use the Gateway-compatible pIX-HALO in vitro expression vector, 26,27 which has both T7 and SP6…”

Profiling Interactome Networks with the HaloTag‐NAPPA In Situ Protein Array

“…Protein microarrays have been used to investigate biochemical phenomena such as protein‐protein interactions (PPIs), protein‐DNA interactions, or protein‐chemical interactions for more than 15 years (Zhu et al., ). The various types of protein microarrays include conventional spotting protein arrays (Zhu et al., ), antibody arrays (Chaga, ; Rivas et al., ), nucleic acid programmable protein assays (NAPPAs; Ramachandran et al., , ; Yazaki et al., ), protein in situ arrays (PISA; He & Taussig, ), and DNA array to protein arrays (DAPA; He et al., ). For the conventional “protein spotting arrays,” it is necessary to prepare purified in vivo ‐expressed proteins and spot the purified proteins on glass slides, which involves considerable cost, time, and labor.…”

“…The recently developed HaloTag‐NAPPA in situ protein arrays consisting of HaloTag, a modified bacterial enzyme protein (Los et al., ), can solve these issues. A higher signal‐to‐noise ratio was shown in HaloTag‐NAPPA compared to that in GST‐based original arrays (Yazaki et al., ). HaloTag can covalently bond to a small chemical ligand molecule, chloroalkane, which is co‐spotted with the ORF plasmid DNA to the array.…”

“…Protein arrays enable screening of thousands of proteins in a single experiment using a panel of probes (Gong et al., ; Lin et al., ; Popescu et al., , ; Ramachandran et al., , ; Yazaki et al., ; Zhu et al., ) for the detection of protein‐protein, protein‐DNA/RNA, and protein‐small molecule interactions and analysis of protein modifications. Here, we describe the protocol for fabricating the HaloTag‐NAPPA protein array (Yazaki et al., ), which we developed based on the nucleic acid programmable protein assay (NAPPA; Ramachandran et al., , ). We modified the original system by fabricating a protein chip spotted with plasmid DNA that is then translated into protein using an in vitro expression (IVX) system.…”

“…We also introduced a high‐affinity capture tag known as the HaloTag (Promega) to immobilize nascent proteins on the array surface. The HaloTag‐NAPPA was applied to detect the protein‐protein interaction for a set of transcription factors (TFs) involved in the plant hormone signaling pathway (Yazaki et al., ), and to contribute to the development of the TF interactome network at the proteome scale. This article provides instructions for HaloTag‐NAPPA construction, protein expression, and analysis of protein‐protein interactions using an open reading frame (ORF) resource.…”