Introduction Oxygen homeostasis is tightly regulated in multicellular organisms by the transcription factor hypoxia-inducible factor- 1 (HIF-1). In response to decreased oxygen levels HIF-1 activates transcription of target genes encoding proteins critical for several developmental and physiological processes including glycolysis and angiogenesis (Semenza, 2002). HIF- 1 is a heterodimeric complex composed of the transcription factors HIF-1α and Arnt. In contrast to Arnt, oxygen levels regulate both the expression and the activity of HIF-1α. At normoxia (21% O 2 ) HIF-1α is rapidly ubiquitylated and degraded by the 26S proteasome (Huang et al., 1998; Kallio et al., 1997; Kallio et al., 1999; Salceda and Caro, 1997) whereas at hypoxia (1% O 2 ) the protein is stabilized. Degradation of HIF-1α is regulated by hydroxylation of specific proline residues that are recognized by the von Hippel-Lindau tumor suppressor protein which is part of an E3 ubiquitin ligase complex (Cockman et al., 2000; Ivan et al., 2001; Jaakkola et al., 2001; Kamura et al., 2000; Masson et al., 2001; Ohh et al., 2000; Pereira et al., 2003; Tanimoto et al., 2000; Yu et al., 2001). At low oxygen levels HIF-1α translocates to the nucleus (Kallio et al., 1998) where the functionally active HIF-1α/Arnt complex activates transcription of target genes after binding to cognate hypoxia-responsive elements (HRE). HIF-1-mediated activation of transcription requires the recruitment of coactivators such as CBP/adenovirus E1A-binding protein p300 (p300) and factors belonging to the steroid receptor coactivator (SRC)/p160 family of proteins (Arany et al., 1996; Carrero et al., 2000; Ebert and Bunn, 1998; Ema et al., 1999; Gu et al., 2001; Kung et al., 2000; Ruas et al., 2002). Hydroxylation of an asparagine residue at normoxia by the factor inhibiting HIF-1α (FIH-1) regulates the transactivation activity of HIF-1α by abrogating the interaction between the C-terminal transativation domain (C-TAD) of HIF-1α and the CH1 domain of the CREB-binding protein (CBP) (Hewitson et al., 2002; Lando et al., 2002a; Lando et al., 2002b; Mahon et al., 2001). CBP/p300 regulate chromatin structure through histone acetylation and interaction with other histone acetyltransferases including P/CAF (Yang et al., 1996) and are known to acetylate other proteins involved in the regulation of transcription (Sterner and Berger, 2000). The p160/SRC proteins, constituting a family of coactivators, have been identified mainly as nuclear hormone receptor-interacting proteins (Xu and Li, 2003). SRCs have been proposed to function as nuclear hormone receptor coactivators by recruiting histone acetyltransferases such as CBP/p300 and P/CAF (Li et al., 2000; Spencer et al., 1997) and histone methyltransferases such as coactivator-associated arginine methyltransferase-1 (CARM1) and protein arginine N-methyltransferase-1 (PRMT1) (Chen et al., 1999; Koh et al., 2001). Previous reports have shown that the function of both the N- terminal (N-TAD) and C-terminal (C-TAD) transactivation domains of HIF-1α can be enhanced by CBP and SRC-1 in 301 The hypoxia-inducible factor-1 (HIF-1) is a key regulator of oxygen homeostasis in the cell. We have previously shown that HIF-1α and the transcriptional coactivator CBP colocalize in accumulation foci within the nucleus of hypoxic cells. In our further exploration of the hypoxia-dependent regulation of HIF-1α function by transcriptional coactivators we observed that coexpression of SRC-1 (another important coactivator of the hypoxia response) and HIF-1α did not change the individual characteristic nuclear distribution patterns. Colocalization of both these proteins proved to be mediated by CBP. Biochemical assays showed that depletion of CBP from cell extracts abrogated interaction between SRC-1 and HIF-1α. Thus, in contrast to the current model for the assembly of complexes between nuclear hormone receptors and coactivators, the present data suggest that it is CBP that recruits SRC-1 to HIF-1α in hypoxic cells. We also observed that CBP, HIF-1α/Arnt and HIF-1α/CBP accumulation foci partially overlap with the hyperphosphorylated form of RNA polymerase II, and that CBP had a stabilizing effect on the formation of the complex between HIF-1α and its DNA-binding partner, Arnt. In conclusion, CBP plays an important role as a mediator of HIF-1α/Arnt/CBP/SRC-1 complex formation, coordinating the temporally and hierarchically regulated intranuclear traffic of HIF-1α and associated cofactors in signal transduction in hypoxic cells. Key words: Hypoxia-inducible factor-1, CREB-binding protein, Steroid receptor coactivator-1, Intranuclear distribution, Cyan/yellow fluorescent protein Summary Role of CBP in regulating HIF-1-mediated activation of transcription Jorge L. Ruas, Lorenz Poellinger* and Teresa Pereira Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden *Author for correspondence (e-mail: lorenz.poellinger@cmb.ki.se) Accepted 29 October 2004 Journal of Cell Science 118, 301-311 Published by The Company of Biologists 2005 doi:10.1242/jcs.01617 Research Article Journal฀of฀Cell฀Science