Contribution of the Per/Arnt/Sim (PAS) Domains to DNA Binding by the Basic Helix-Loop-Helix PAS Transcriptional Regulators

Anne Chapman-Smith,Jodi K Lutwyche,Murray L Whitelaw

doi:10.1074/jbc.m310041200

Anne Chapman-Smith, Jodi K Lutwyche + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m310041200

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Abstract

The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Here, we have analyzed purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1alpha and evaluated the contribution of the PAS domains to DNA binding in vitro. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1alpha with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences required the PAS A domains and exhibited KD values of 0.4 nM and approximately 50 nM, respectively. In contrast, the presence of the PAS domains of Arnt had little effect on DNA binding by Arnt homodimers, and these bound DNA with a KD of 45 nM. In the case of the DR, both high affinity DNA binding and dimer stability were specific to its native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generated a destabilized protein that bound DNA poorly.

Highlights

The basic helix-loop-helix1 family of transcriptional regulators is characterized by a highly conserved basic DNA binding sequence adjacent to a helix-loop-helix dimerization ʈ Supported by the Australian Research Council through the Special Research Centre for the Molecular Genetics of Development
Dimerization among basic helix-loop-helix (bHLH).PAS proteins is regulated by the PAS region, which controls the specificity of partner choice such that hypoxia inducible factors (HIF)-␣ and dioxin (aryl hydrocarbon) receptor (DR) must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes
Expression and Purification of Functional DNA-binding Proteins—Proteins comprising either the bHLH or bHLH.PAS domains of Arnt, the DR and HIF-1␣ were expressed in E. coli as fusions to TrxH6 or glutathione S-transferase (GST)

Summary

Introduction

The basic helix-loop-helix (bHLH)1 family of transcriptional regulators is characterized by a highly conserved basic DNA binding sequence adjacent to a helix-loop-helix dimerization ʈ Supported by the Australian Research Council through the Special Research Centre for the Molecular Genetics of Development. Both Arnt PAS-containing constructs and the Arnt TrxH6-bHLH domain alone formed a complex with E-box DNA in EMSAs, which could be depleted with anti-Arnt antibody (Fig. 2a), and this DNA binding activity was specific for the E-box sequence as no DNA binding was observed when these proteins were incubated with the XRE (Fig. 2c, lanes 1–3) or the HRE (Fig. 2d, lane 3) probes.

Results

Conclusion