Investigation of a transcription factor complex and intrinsically disordered proteins

Abstract

LIM domain binding protein 1 (Ldb1) is a chromatin looping factor that forms part of a transcriptional ‘pentameric complex’. Ldb1 contains two domains that are essential for looping: a self-association domain, and a LIM interaction domain (LID) that binds LIM proteins such as Lmo2, which in turn binds to DNA binding transcription factors, Tal1, E2a and Gata-1. It was proposed that the Gata-1 binding protein FOG1 could bind to the pentameric complex. GFP-tagged FOG1 was shown to bind the complex by multi angle laser light scattering, providing a mechanism by which the intrinsically weak Gata-1/FOG1 interaction is bolstered through binding to other units of the pentameric complex. Little is known about proteins that are distantly related to mammalian Ldb1. Two such proteins, Ldb1 from C. elegans and Adn1 from S. pombe were expressed in bacteria. They had generally poor solubility, but use of a maltose binding protein tag promoted solubility, and different expression systems may enable their further study. Ldb1 was reported to bind the E3 ubiquitin ligase protein, RLIM. No interaction could be detected between these proteins by yeast two-hybrid analysis using truncated or full length proteins. The interaction was detected in mammalian cells using FLAG pull-down experiments, but truncation mutants of these proteins could not be expressed. RLIM has high levels of predicted disorder, which may contribute to its degradation in both cell types. An assay was developed in which dimerization domains could stabilise disordered binding regions. Constructs containing GST, a coiled-coil domain of CtIP, or the leucine zipper of GCN4, were tethered to a test peptide and assayed for binding in yeast two-hybrid assays. The domains from CtIP and/or GCN4 allowed the interaction to be detected. Although the assay could not detect an interaction between RLIM and Ldb1, it shows promise for detecting interactions for disordered proteins, and can be adapted to different expression systems.