The Basic Helix-Loop-Helix Domain of the Aryl Hydrocarbon Receptor Nuclear Transporter (ARNT) Can Oligomerize and Bind E-box DNA Specifically

Abstract

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a basic helix-loop-helix (bHLH) protein that contains a Per-Arnt-Sim (PAS) domain. ARNT heterodimerizes in vivo with other bHLH PAS proteins to regulate a number of cellular activities, but a physiological role for ARNT homodimers has not yet been established. Moreover, no rigorous studies have been done to characterize the biochemical properties of the bHLH domain of ARNT that would address this issue. To begin this characterization, we chemically synthesized a 56-residue peptide encompassing the bHLH domain of ARNT (residues 90 -145). In the absence of DNA, the ARNT-bHLH peptide can form homodimers in lower ionic strength, as evidenced by dynamic light scattering analysis, and can bind E-box DNA (CACGTG) with high specificity and affinity, as determined by fluorescence anisotropy. Dimers and tetramers of ARNT-bHLH are observed bound to DNA in equilibrium sedimentation and dynamic light scattering experiments. The homodimeric peptide also undergoes a coil-to-helix transition upon E-box DNA binding. Peptide oligomerization and DNA affinity are strongly influenced by ionic strength. These biochemical and biophysical studies on the ARNT-bHLH reveal its inherent ability to form homodimers at concentrations supporting a physiological function and underscore the significant biochemical differences among the bHLH superfamily.

Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development

“…This domain is found in over one-third of Arabidopsis bHLH TF family members (Supplemental Data Set 3). The bHLH transcription factors usually function in dimer or tetramer form or by forming a complex with other TF family members (Ellenberger et al, 1994;Ma et al, 1994;Huffman et al, 2001;Li, 2014;Chang et al, 2015); often the bHLH motif is required for dimerization. In this study, we demonstrated that the plantspecific BIF domain is also important for the dimerization and the normal function of DYT1.…”

Unique and Overlapping Transcriptional Roles of Arylhydrocarbon Receptor Nuclear Translocator (Arnt) and Arnt2 in Xenobiotic and Hypoxic Responses

“…Homodimerization of Arnt and Arnt H378P-Arnt can form a heterodimer with AhR and HIF1␣, but it also homodimerizes and binds E-box sequences (25,34,35). We investigated the ability of ArntH378P to homodimerize with either WT or mutant Arnt, using the mammalian two-hybrid assay with GAL4DBD-Arnt-bHLHPAS or GAL4DBD-ArntH378P-bHL-HPAS as bait (Fig.…”

HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endothelia

“…Complexes of the A13 DNA binding domain peptide and DNA were assessed for their oligomerization state using sedimentation equilibrium ultracentrifugation on a Beckman Coulter XL-1A Protein Analysis System as described (Huffman et al, 2001). A self-annealing fluorescein-labeled oligonucleotide, 5Ј-6-FAM-CCCATAAACCCCCCCGGTT-TATGGG-3Ј (5 M) was combined with the A13 DNA binding peptide (6 M to 10 M) in a buffer containing 80 mM KCl, 10 mM MgCl 2 , 0.2 mM EDTA, 1 mM dithiothreitol, and 20 mM Tris.HCl pH 7.8.…”