Regulation of gene expression by hypoxia: integration of the HIF-transduced hypoxic signal at the hypoxia-responsive element

Abstract

Cells experiencing lowered O(2) levels (hypoxia) undergo a variety of biological responses in order to adapt to these unfavorable conditions. The master switch, orchestrating the cellular response to low O(2) levels, is the transcription factor, termed hypoxia-inducible factor (HIF). The alpha subunits of HIF are regulated by 2-oxoglutarate-dependent oxygenases that, in the presence of O(2), hydroxylate specific prolyl and asparaginyl residues of HIF-alpha, inducing its proteasome-dependent degradation and repression of transcriptional activity, respectively. Hypoxia inhibits oxygenases, stabilized HIF-alpha translocates to the nucleus, dimerizes with HIF-beta, recruits the coactivators p300/CBP, and induces expression of its transcriptional targets via binding to hypoxia-responsive elements (HREs). HREs are composite regulatory elements, comprising a conserved HIF-binding sequence and a highly variable flanking sequence that modulates the transcriptional response. In summary, the transcriptional response of a cell is the end product of two major functions. The first (trans-acting) is the level of activation of the HIF pathway that depends on regulation of stability and transcriptional activity of the HIF-alpha. The second (cis-acting) comprises the characteristics of endogenous HREs that are determined by the availability of transcription factors cooperating with HIF and/or individual HIF-alpha isoforms.

Figure 1. HIF-1α: schematic outline of the domain structure, function of individual domains, and the sites of post-translational modifications critically affecting its function

bHLH, basic loop-helix-loop; CAD, C-terminal activation domain; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; FIH-1, factor inhibiting HIF 1; NAD, N-terminal activation domain; NLS, nuclear localization signal; ODDD, O₂ –dependent degradation domain; PAS, PER-ARNT-SIM; PHDs, prolyl hydroxylases.

Figure 2. The outline of regulation of stability and transcriptional activity of HIF-α

In the presence of O₂ and cofactors Fe²⁺ and 2OG, PHDs hydroxylate HIF-α, allowing its recognition by the E3 complex that is followed by E2/E1-mediated ubiquitylation and degradation in the 26S proteasome. FIH-1-mediated N-hydroxylation prevents recruitment of p300/CBP transcriptional coactivators. In the absence of O₂, PHDs and FIH-1 are inactivated, non-hydroxylated HIF-α translocates to the nucleus, dimerizes with HIF-β, recruits p300/CBP, and induces the expression of its target genes via binding to the HRE. BTM, basic transcriptional machinery; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; FIH-1, factor inhibiting HIF-1; HRE, hypoxia-response element; 2OG, 2-oxoglutarate; PHDs, prolyl hydroxylases; Ub, ubiquitin; VHL, von Hippel-Lindau protein.

Figure 3

(A) Numbering of the nucleotide positions in HBS (the core sequence in bold); (B). Schematic outline of HBS and HAS in the EPO HRE; (C) Schematic outline of the two fundamental arrangements of HBSs present in the mLDHA (antiparallel) and mPGK-1 (parallel) HRE. Arrows indicate the orientation of HBSs.