Hox Transcription Factor Ultrabithorax Ib Physically and Genetically Interacts with Disconnected Interacting Protein 1, a Double-stranded RNA-binding Protein

Abstract

The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0 -12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx⅐DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx⅐DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNAbinding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either singlestranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated.

Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila

“…This motif was first identified in the developmentally essential gene Staufen of Drosophila melanogaster and has since been recognized to be encoded in the genomes in all three domains of living organisms, as well as in viruses (63; reviewed in references 20 and 67). DSRM proteins commonly act in developmental pathways (e.g., RNA localization by the Staufen family and developmental transcriptional regulation by the DIP1 family) (5,18,62,68) but also have ubiquitous roles in transcriptional and translational regulation (e.g., PKR family and PKR-associated proteins) (26,45,55,58). Proteins vital for RNA interference (RNAi) also contain DSRMs.…”

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

“…However, a subset of Hox-regulated enhancers lack sites for known Hox partners. Thus, several laboratories, including ours, have been exploring the hypothesis that amino acid sequences outside the homeodomain contribute to differences in binding site selection by Hox proteins dur-ing animal development (6,15,16,20,(25)(26)(27)(28). These nonhomeodomain sequences vary significantly between Hox proteins (supplemental Fig.…”

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

“…3D). In addition, Ubx binds other transcription factors that have their own DNA-binding sites near those of Ubx target DNA sequences (45,68). This arrangement provides opportunities for creating combinatorial loops that are sensitive to cellular conditions and allow response to cell-signaling stimuli.…”

“…phosphorylation) (38,82,83), and a number of protein partners (Fig. 4C) (31,45,68). These regulatory mechanisms are frequently used to diversify the functions of transcription factors (84).…”

“…The disordered regions also mediate Ubx binding to a variety of heteroprotein partners, a critical element in Hox protein function (45,68,69,86). Hox proteins bind to components of the transcription machinery (45,87), as well as to other specific transcription factors, to facilitate Hox regulation of the correct subset of genes in different tissues (Fig.…”