**2. ARNT/HIF-1β**

#### **2.1 ARNT/HIF-1β structure and function**

ARNT/HIF-1β belongs to a group of transcription factors, known as the basic helix loop helix - PER/ARNT/Sim (bHLH-PAS) family, which has a characteristic N-terminal bHLH motif for DNA binding, a central PAS domain which facilitates heterodimerization and a Cterminal transactivation domain for the recruitment of transcriptional coactivators such as CBP/p300 (Jain et al., 1994; Kobayashi et al., 1997). Recent evidence suggest that the PAS domain may also provide an additional binding site for coactivators and thereby recruiting them in a step necessary for transcriptional responses to hypoxia (Partch & Gardener, 2011). ARNT/HIF-1β acts as a common binding partner for most of the bHLH-PAS family of transcription factors and bind specific DNA sequences in the regulatory regions of the responsive genes. The half-site for ARNT/HIF-1β is on the 3' side of the 5'-GTG-3' recognition sequence. The sequence of the other half of the binding site depends upon the identity of the ARNT/HIF-1β dimerization partner (Swanson et al., 1995). DNA binding of ARNT/HIF-1β is mediated by its bHLH region and may also involve the PAS region. Dimerization between ARNT/HIF-1β and other bHLH-PAS proteins is mediated by their bHLH and PAS regions (Jiang et al., 1996, Lindebro et al., 1995). The human ARNT/HIF-1β gene is about 65 Kb in size, has 22 exons and is well conserved on an evolutionary scale (Scheel & Schrenk, 2000).

ARNT/HIF-1β was originally cloned as a factor required for the activity of the aryl hydrocarbon receptor (AhR). AhR induces a transcriptional response to various environmental pollutants, such as polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds (Reyes et al., 1992). ARNT/HIF-1β was also identified as the β-subunit of a heterodimeric transcription factor, hypoxia-inducible factor 1α (HIF-1α) (Wang et al., 1995 (a)). Similar to HIF-1α, ARNT/HIF-1β gene expression and protein levels are significantly increased under hypoxic conditions suggesting that this gene plays an important role in the transcriptional response to low oxygen tension (Wang et al., 1995 (b)). Consistent with this idea, it has been shown that ARNT/HIF-1β is essential for the hypoxic induction of vascular endothelial growth factor (VEGF) and the glycolytic enzymes aldolase A (ALDO) and phosphoglycerate kinase (PGK) in a mouse hepatoma (Hepa 1c1c7) cell line (Li et al., 1996; Salceda et al., 1996). Unlike HIF-1α, which is exclusively expressed under hypoxic conditions, ARNT/HIF-1β is constitutively expressed in a number of tissues, such as the brain, heart, kidney, muscles, thymus, retina, olfactory epithelium and beta-cells of pancreas (Hirose et al., 1996).

#### **2.2 ARNT/HIF-1β localization, binding partners, mechanism of action and lessons from knockout animals**

ARNT/HIF-1β is a nuclear protein in most cell types, although it may also be located in the cytosol, particularly during embryogenesis. Studies conducted by Holmes and Pollenz (1997) in hepatic and non-hepatic cell lines derived from rat, mouse, human, and canine tissues confirm ARNT/HIF-1β as a nuclear transcription factor and showed that its physical interaction with DNA requires entry into the nucleus.

ARNT/HIF-1β serves as an obligatory binding partner for a number of other bHLH-PAS proteins, whose activity is modulated either by exogenous chemicals (AhR), hypoxia (HIF-1α, HIF-2α and HIF-3α), or which show tissue-specific expression pattern (e.g. SIM-1) (Salceda et al., 1996; Swanson et., 1995; Woods & Whitelaw, 2002). In addition to forming heterodimers, ARNT/HIF-1β appears to be capable of forming homodimers and bind to an E-box sequence 5'-CACGTG-3' (Antonsson et al., 1995). It was also shown that ARNT/HIF-1β homodimer regulates the transcription of murine cytochrome P450 (Cyp) 2a5 gene

motif for DNA binding, a central PAS domain which facilitates heterodimerization and a Cterminal transactivation domain for the recruitment of transcriptional coactivators such as CBP/p300 (Jain et al., 1994; Kobayashi et al., 1997). Recent evidence suggest that the PAS domain may also provide an additional binding site for coactivators and thereby recruiting them in a step necessary for transcriptional responses to hypoxia (Partch & Gardener, 2011). ARNT/HIF-1β acts as a common binding partner for most of the bHLH-PAS family of transcription factors and bind specific DNA sequences in the regulatory regions of the responsive genes. The half-site for ARNT/HIF-1β is on the 3' side of the 5'-GTG-3' recognition sequence. The sequence of the other half of the binding site depends upon the identity of the ARNT/HIF-1β dimerization partner (Swanson et al., 1995). DNA binding of ARNT/HIF-1β is mediated by its bHLH region and may also involve the PAS region. Dimerization between ARNT/HIF-1β and other bHLH-PAS proteins is mediated by their bHLH and PAS regions (Jiang et al., 1996, Lindebro et al., 1995). The human ARNT/HIF-1β gene is about 65 Kb in size, has 22 exons and is well conserved on an evolutionary scale

ARNT/HIF-1β was originally cloned as a factor required for the activity of the aryl hydrocarbon receptor (AhR). AhR induces a transcriptional response to various environmental pollutants, such as polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds (Reyes et al., 1992). ARNT/HIF-1β was also identified as the β-subunit of a heterodimeric transcription factor, hypoxia-inducible factor 1α (HIF-1α) (Wang et al., 1995 (a)). Similar to HIF-1α, ARNT/HIF-1β gene expression and protein levels are significantly increased under hypoxic conditions suggesting that this gene plays an important role in the transcriptional response to low oxygen tension (Wang et al., 1995 (b)). Consistent with this idea, it has been shown that ARNT/HIF-1β is essential for the hypoxic induction of vascular endothelial growth factor (VEGF) and the glycolytic enzymes aldolase A (ALDO) and phosphoglycerate kinase (PGK) in a mouse hepatoma (Hepa 1c1c7) cell line (Li et al., 1996; Salceda et al., 1996). Unlike HIF-1α, which is exclusively expressed under hypoxic conditions, ARNT/HIF-1β is constitutively expressed in a number of tissues, such as the brain, heart, kidney, muscles, thymus, retina, olfactory epithelium and beta-cells

**2.2 ARNT/HIF-1β localization, binding partners, mechanism of action and lessons** 

ARNT/HIF-1β is a nuclear protein in most cell types, although it may also be located in the cytosol, particularly during embryogenesis. Studies conducted by Holmes and Pollenz (1997) in hepatic and non-hepatic cell lines derived from rat, mouse, human, and canine tissues confirm ARNT/HIF-1β as a nuclear transcription factor and showed that its physical

ARNT/HIF-1β serves as an obligatory binding partner for a number of other bHLH-PAS proteins, whose activity is modulated either by exogenous chemicals (AhR), hypoxia (HIF-1α, HIF-2α and HIF-3α), or which show tissue-specific expression pattern (e.g. SIM-1) (Salceda et al., 1996; Swanson et., 1995; Woods & Whitelaw, 2002). In addition to forming heterodimers, ARNT/HIF-1β appears to be capable of forming homodimers and bind to an E-box sequence 5'-CACGTG-3' (Antonsson et al., 1995). It was also shown that ARNT/HIF-1β homodimer regulates the transcription of murine cytochrome P450 (Cyp) 2a5 gene

(Scheel & Schrenk, 2000).

of pancreas (Hirose et al., 1996).

interaction with DNA requires entry into the nucleus.

**from knockout animals** 

through a palindromic E-box element in the 5' regulatory region of Cyp2a5 gene in primary hepatocytes (Arpiainen et al., 2007). Two ARNT-related genes, ARNT-2 and ARNT-3 (also called BMAL-1 or MOP3) have been identified. ARNT-2 is more restricted in expression than ARNT/HIF-1β, but appears to dimerize with the same partner proteins as ARNT/HIF-1β (Hirose et al., 1996). ARNT-3 appears to have different dimerization potential than ARNT/HIF-1β (Ikeda & Nomura, 1997). The transactivation potential of ARNT/HIF-1β is not only determined through the recruitment of transcriptional cofactors, but also by signaling input from several protein kinases, such as PKC (Long et al., 1999).

The ARNT/HIF-1β/AhR heterodimer activates transcription of several genes involved in metabolism of foreign chemicals, including CYP1A1, CYP1B1, and NADP(H):oxidoreductase (NQO1) (Sogawa & Kuriayama, 1997; Beischlag et al., 2008). Transcriptional activation of these genes depends upon prior binding of AhR to xenobiotic ligands, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (dioxin) and benzopyrene. The ARNT/HIF-1β/AhR heterodimer and ARNT/HIF-1β can have an impact on estrogen receptor (ER) activity. ARNT/HIF-1β interacts and functions as a potent coactivator of both ER-α and ER-β dependent transcription and it is believed that the C-terminal domain of ARNT/HIF-1β is essential for the transcriptional enhancement of ER activity (Lim et al., 2011;Rüegg et al., 2008).

ARNT/HIF-1β/HIF-1α heterodimer activity is primarily regulated by HIF-1α protein stability. Under normoxia, HIF-1α is hydroxylated by an oxygen requiring enzyme, prolyl hydroxylase (PHD), which is then targeted for ubiquitination by the E3 ubiquitin ligase, followed by binding to von Hippel-Lindau tumor suppressor (VHL) which leads to degradation of HIF-1α by the proteasome pathway. Conversely, under hypoxic conditions, a lack of oxygen inhibits hydroxylation, leading to stabilization of the HIF-1α protein and translocation of HIF-1α from the cytoplasm to the nucleus. In the nucleus, heterodimerization of HIF-1α with ARNT/HIF-1β is followed by binding to hypoxia response elements (HRE) in the promoter region of the target genes (Fedele et al., 2002) (Figure 2). Like HIF-1α, HIF-2α and 3α are stabilized by hypoxia and hypoglycemia, and activate transcription of genes involved in adapting to these adverse conditions, including the genes for erythropoietin (EPO), VEGF, and a number of enzymes of glycolysis including ALDO, phosphofructokinase (PFK) and lactate dehydrogenase (LDH) (Maltepe et al., 1997; Fraisl et al., 2009; Fedele et al., 2002). These studies suggest ARNT/HIF-1β is a central player in a number of signaling pathways and alterations in its activity can have serious impact on cellular responses to hypoxia, dioxin response and estrogen signaling in mammalian cells.

Observations from the ARNT/HIF-1β conditional knockout mice and whole body knockout mice have provided a wealth of information regarding the functional significance of this transcription factor in mammalian cells. Results obtained from the ARNT/HIF-1β null mice suggest that it plays a central role in embryonic development and physiological homeostasis as these mice are embryonic lethal due to severe defects in angiogenesis and placental development (Maltepe et al., 1997; Kozak et al., 1997). Data obtained from tissue specific ARNT/HIF-1β knockout mice demonstrates that disruption of ARNT/HIF-1β expression in liver and heart results in loss of AhR-stimulated gene transcription and that ARNT/HIF-1β is key to AhR function in these two mammalian tissues. It was also observed that ARNT/HIF-1β affects HIF-1α mediated target gene expression as several key genes including the expression of heme-oxygenase and glucose transporter-1 mRNA was abolished after treatment with CoCl2, an agent that is thought to mimic hypoxia (Tomita et al., 2000).

Fig. 2. Overview of gene regulation by ARNT/HIF-1β/HIF-1α complex in mammalian cells under normoxic and hypoxic conditions. In normoxic conditions, HIF-1α protein undergoes oxygen dependent hydroxylation by prolyl hydroxylases (PHD) and the hydroxylation site is recognized by pVHL, which targets the protein for ubiquitination by ubiquitin ligase, followed by degradation through ubiquitin proteasome pathway. During hypoxia, HIF-1α protein is not targeted for degradation and can translocate to the nucleus, where it heterodimerizes with ARNT/HIF-1β to form a stable transcriptional complex. The ARNT/HIF-1β/HIF-1α heterodimer then binds to the hypoxia response element (HRE) of target genes.
