Development and application of a DNA microarray-based yeast two-hybrid system

Abstract

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

“…First, Epi-Decoder requires a library of tagged proteins. For budding yeast, several tagged protein libraries are already available in addition to the carboxy-terminal TAP-tag library used here [55][56][57] . This will allow for extending the current Epi-Decoder analyses to a nearly complete coverage of the proteome as well as to alternative (amino-terminal) tags, together enabling an unprecedented deep analysis of chromatin proteomes.…”

DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy

“…Y2H expression plasmids were generated with the Gateway technology (Invitrogen) as recommended by the manufacturer. Y2H vectors were: pDONR221 (Invitrogen), used as entry vector, and pBTM116-D9 and pACT4-DM (Goehler et al, 2004;Stelzl et al, 2005;Suter et al, 2013), as bait and prey destination vectors, respectively. Additionally, pFLAG-CMV-D11 (FLAG-tagged destination vector) was used for co-IP experiments (Invitrogen).…”

Next-Generation Sequencing for Binary Protein–Protein Interactions

“…Notably, the use of DNA microarrays for parallel identification of Y2H screening results was recognized early on ( Cho et al, 1998 ). More recently, a microarray-Y2H screening and scoring system was introduced and applied to identify interaction partners of huntingtin and ataxin-1, two important determinants for neurodegenerative diseases ( Suter et al, 2013 ). Using the Qi-Sampler repeat sampling tool ( Fontaine et al, 2011 ), microarray-Y2H results were benchmarked against sets of known positives (golden sets) and other gene sets for statistical enrichments.…”