The basic-helix–loop–helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors

The basic-helix–loop–helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors

^*Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611; and ^†The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706

Communicated by James A. Miller, University of Wisconsin, Madison, WI (received for review January 26, 1998)

Abstract

We report that MOP3 is a general dimerization partner for a subset of the basic-helix–loop–helix (bHLH)-PER–ARNT–SIM (PAS) superfamily of transcriptional regulators. We demonstrated that MOP3 interacts with MOP4, CLOCK, hypoxia-inducible factor 1α (HIF1α), and HIF2α. A DNA selection protocol revealed that the MOP3-MOP4 heterodimer bound a CACGTGA-containing DNA element. Transient transfection experiments demonstrated that the MOP3-MOP4 and MOP3-CLOCK complexes bound this element in COS-1 cells and drove transcription from a linked luciferase reporter gene. We also deduced the high-affinity DNA binding sites for MOP3-HIF1α complex (TACGTGA) and used transient transfection experiments to demonstrate that the MOP3-HIF1α and MOP3-HIF2α heterodimers bound this element, drove transcription, and responded to cellular hypoxia. Finally, we found that MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules in vivo.

Footnotes

↵ ‡ To whom reprint requests should be addressed. e-mail: bradfield{at}oncology.wisc.edu.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF044288).
↵ § Nomenclature of bHLH-PAS proteins: We now refer to MOP1 as HIF1α and MOP2 as HIF2α (6). MOP2 is also referred to as EPAS1, HRF, and HLF by other laboratories (12, 14, 35). MOP3 is also referred to as BMAL1 (13). MOP4 is also referred to as NPAS2 and MOP5 as NPAS1 (11).
ABBREVIATIONS:

PAS,

PER–ARNT–SIM homology domain;

bHLH,

basic-helix–loop–helix domain;

AHR,

aryl hydrocarbon receptor;

ARNT,

aryl hydrocarbon receptor nuclear translocator;

HIF1α,

hypoxia-inducible factor 1α;

PER,

product of the Drosophila period gene;

RPA,

ribonuclease protection assay;

M34,

the response element bound by the MOP3-MOP4 complex;

M13,

the response element bound by the HIF1α-MOP3 complex;

CLOCK,

product of the Clock locus in mice;

E-box,

an enhancer element defined as CANNTG