Review
The mammalian basic helix–loop–helix/PAS family of transcriptional regulators

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Abstract

Basic helix–loop–helix (bHLH)/PAS proteins are critical regulators of gene expression networks underlying many essential physiological and developmental processes. These include transcriptional responses to environmental pollutants and low oxygen tension, mediated by the aryl hydrocarbon (Dioxin) receptor and hypoxia inducible factors (HIF), respectively, and controlling aspects of neural development, mediated by the single minded (SIM) proteins. bHLH proteins must dimerise to form functional DNA binding complexes and bHLH/PAS proteins are distinguished from other members of the broader bHLH superfamily by the dimerisation specificity conferred by their PAS homology domains. bHLH/PAS proteins tend to be ubiquitous, latent signal-regulated transcription factors that often recognise variant forms of the classic E-box enhancer sequence bound by other bHLH proteins. Two closely related forms of each of the hypoxia inducible factors α and single minded proteins and the general partner protein, aryl hydrocarbon receptor nuclear translocator (ARNT), are present in many cell types. Despite high sequence conservation within their DNA binding and dimerisation domains, and having very similar DNA recognition specificities, the homologues are functionally non-redundant and biologically essential. While the mechanisms controlling partner choice and target gene activation that determine this functional specificity are poorly understood, interactions mediated by the PAS domains are essential. Information on structures and protein/protein interactions for members of the steroid hormone/nuclear receptor superfamily has contributed to our understanding of the way these receptors function and assisted the development of highly specific agonists and antagonists. Similarly, it is anticipated that developing a detailed mechanistic and structural understanding of bHLH/PAS proteins will ultimately facilitate drug design.

Introduction

The bHLH transcriptional regulators are critical for controlling the activity of gene expression networks in many fundamental biological processes, such as early cell determination and differentiation, cell cycle maintenance, and homeostasis or stress response pathways (Massari & Murre, 2000). This extensive protein family is characterised by a basic DNA binding region adjacent to a helix–loop–helix dimerisation region, both of which are required for formation of functional DNA binding complexes.

There are three main sub-families of bHLH proteins: (a) those containing only the bHLH domain; and those where the bHLH domain is contiguous with a second dimerisation domain, either (b) the leucine zipper (Zip) or (c) the PER/aryl hydrocarbon receptor nuclear translocator (ARNT)/single minded (SIM) (PAS) homology domain (Fig. 1). The first group includes mammalian proteins involved in myogenesis (MyoD, myogenin and their partner E47) neurogenesis (NeuroD, neurogenin) or haemopoiesis (SCL) (Massari & Murre, 2000). The addition of the leucine zipper is observed in members of the Myc/Max/Mad network of transcription factors (Lüscher, 2001). Both the bHLH and bHLH/Zip proteins recognise the classic E-box core enhancer sequence (CANNTG) in their target genes and generally exhibit constitutive activity corresponding to restricted tissue or developmental expression patterns (Massari & Murre, 2000). In contrast, the bHLH/PAS proteins tend to be ubiquitous, latent transcription factors whose activity is signal regulated. Furthermore, bHLH/PAS heterodimers are distinguished by recognising DNA sequences which often diverge from the prototypical E-box (Crews, 1998, Crews & Fan, 1999, Taylor & Zhulin, 1999). The precision of gene control by bHLH proteins involves interactions between elements of the bHLH domain, contiguous Zip or PAS domain, intervening sequences and transactivation domains, in a manner not well understood (Crews, 1998, Crews & Fan, 1999, Massari & Murre, 2000, Taylor & Zhulin, 1999).

Many aspects of the functional control of bHLH/PAS proteins are analogous to the nuclear receptor signalling proteins such as the Vitamin D and retinoic acid receptors. Both systems combine a specific receptor with a generic partner protein, and family members can be grouped into two classes (Fig. 1). Class I bHLH/PAS factors neither homodimerise nor heterodimerise with other Class I factors, and include the aryl hydrocarbon receptor (AhR), the hypoxia inducible factors (HIF; HIF-1α, HIF-2α, and HIF-3α) and single minded proteins (SIM1 and SIM2) (Ema et al., 1996; Lindebro et al., 1995, Wang et al., 1995). To form active transcription factor complexes they must dimerise with a Class II bHLH/PAS factor, which promiscuously homo- and heterodimerise. The best characterised Class II protein is the ubiquitous ARNT (Hoffman et al., 1991). Other members of this class include the tissue restricted ARNT2, and the circadian rhythm proteins BMAL1 and BMAL2 (Hirose et al., 1996, Ikeda & Nomura, 1997; Okano, Sasaki, & Fukada, 2001). The bHLH/PAS proteins that function in circadian rhythm maintenance have been recently reviewed by Panda and co-workers (Panda, Hogenesch, & Kay, 2002), and will not be discussed here.

Section snippets

PAS signalling

The PAS region consists of two adjacent degenerate repeats of ∼130 amino acids, PAS A and PAS B. The domain is an ancient signalling device conserved through evolution, having been identified in proteins throughout the animal kingdom, in bacteria, fungi and yeast in addition to mammals and flies, where the most commonly studied bHLH/PAS proteins originate. Signals mediated by PAS domains include redox state, hypoglycaemia, oxygen balance and xenobiotic metabolism, and many bacteria contain

The aryl hydrocarbon receptor

Dioxin (2,3,7,8-tetrachlorodibenzo-ρ-dioxin, TCDD) and structurally related halogenated aromatic hydrocarbons are members of a class of environmental pollutants. Exposure to TCDD and similar compounds results in a variety of biochemical and toxic responses in animal models including severe wasting syndrome, epidermal hyperplasia and metaplasia, tumour promotion and thymic involution (Poland & Knutson, 1982, Safe, 1990).

On a molecular level, responses to TCDD and related chemicals are mediated

Hypoxia inducible factors

The ability to maintain O2 homeostasis is essential for survival of mammals. The hyperoxic state, or high O2 tension, can result in the generation of reactive oxygen intermediates and potentially lethal damage to membranes and DNA. The hypoxic state, or low O2 tension, can result in levels of ATP insufficient to maintain essential cellular functions. The hypoxic state occurs in a number of medical conditions, such as cancer and ischemias, inspiring research into understanding the cellular

Single minded proteins

The first SIM protein identified was from Drosophila melanogaster. dSIM is proposed to act as a master regulator of midline cell development in Drosophila and is required for all known developmental stages of the central nervous system midline cell lineage (Nambu, Lewis, Wharton, & Crews, 1993). dSIM contains a C-terminal transactivation domain, and acts with partner factor dARNT (also known as Tango) as a transcriptional activator (Sonnenfeld et al., 1997).

The murine homologues, mSIM1 and

ARNT

ARNT acts as the general partner factor for members of the bHLH/PAS family of proteins (Fig. 4). ARNT was cloned as the factor required to rescue the mutant phenotype of mouse hepatoma Hepa c4 cells (Hoffman et al., 1991). Upon treatment of Hepa c4 cells with a known AhR ligand, subcellular fractionation experiments found the AhR in the cytoplasm, as opposed to the nucleus in ligand-treated, non-mutant Hepa cells. Transfection of ARNT into Hepa c4 cells resulted in the formation of a

Mechanism of action of the bHLH/PAS proteins

While the function of the PAS domain is not fully understood, it is clear that it forms at least a dimerisation interface, limiting partner selection to other PAS containing proteins. In addition, the PAS domain confers specificity of partner choice within the bHLH/PAS family, as for example, the isolated bHLH domain of the AhR is able to homodimerise, whereas dimerisation is restricted to ARNT by the addition of the adjacent PAS A domain (Pongratz et al., 1998). There is at present little

PAS structure and function

The recent determination of the structures of several PAS proteins shows the existence of a highly conserved PAS structural motif despite relatively low sequence homology (Taylor & Zhulin, 1999). The bacterial light sensor, Photoactive Yellow Protein (PYP; (Borgstahl, Williams, & Getzoff, 1995)), the PAS domain of the human eag-related gene K+ channel (Morais Cabral et al., 1998) and the oxygen sensor FixL from Rhizobia (Gong et al., 1998) adopt the same basic fold (Crews & Fan, 1999;

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