We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

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-␣) 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-␣ and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. 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 domain, which allows homo-or heterodimerization among bHLH proteins to form functional DNA binding complexes. Structural studies indicate that the HLH dimer forms a compact four-helical bundle that positions the basic region to facilitate interaction with the DNA recognition sequence (1). There are two subclasses of bHLH proteins in which formation of protein dimers is additionally regulated by an adjacent secondary dimerization domain, either a leucine zipper (Zip) or a Per/Arnt/Sim (PAS) homology domain (2, 3).The PAS region in bHLH.PAS proteins consists of two adjacent PAS domains, degenerate repeats of ϳ130 amino acids, termed PAS A and PAS B (3). PAS domains are not restricted to bHLH DNA-binding proteins and are a widely observed protein-protein interaction and signaling module forming a highly conserved structure, despite having low primary sequence homology (3-5). The minimal PAS domain structure consists of a 5-stranded ␤-sheet with a central ␣-helical PAS core region, often containing a binding site for small ligands, and a helical connector spanning the two halves of the ␤-scaffold (5, 6). Some PAS domains also appear to include a highly variable N-terminal ␣-helical cap, although the function of this structure is not clear (7,8).In vivo, the aryl hydrocarbon nuclear translocator (Arnt) is the common obligate dimerization partner for a number of bHLH.PAS proteins, including the dioxin (or aryl hydrocarbon) receptor (DR), the hypoxia inducible factors (HIF) 1␣ and 2␣, and the Sim proteins (9). The cytoplasmic DR, on binding an activating ligand such as dioxin or structurally related halogenated hydrocarbons within the PAS B domain (10), translocates to the nucleus and heterodimerizes with Arnt to form a functional DNA binding complex (11). The DR/Arnt dimer recognizes the xenobiotic response element (XRE) in enhancers of target genes and promotes transcription of a battery of xenobiotic-metabolizing enzymes. The HIF␣ proteins mediate cellular responses to oxygen. They are rapidly turned over at normoxia whereas under hypoxic stress, HIF␣ is stabilized in a form competent to recruit coactivators (12). Under hypoxia, HIF␣ heterodimerizes with Ar...

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