Transcription of mammalian cytochrome c oxidase subunit IV‐2 is controlled by a novel conserved oxygen responsive element

Abstract

Oxygen sensing and the adaptation to varying oxygen concentrations are fundamental for the survival of species from bacteria to humans. Oxygen regulation can occur at both the intake stage and the usage stage. In higher organisms, which depend largely on aerobic energy metabolism, usage takes place largely at the last step of the mitochondrial respiratory chain, the transfer of electrons from cytochrome c to oxygen, catalyzed by cytochrome c oxidase (CcO; EC 1.9.3.1).In vertebrates, oxygen supply is also regulated via a unique mechanism in the lungs for hypoxic response. Other tissues (and, indeed, the bronchial circulatory system of the lung) react to a hypoxic trigger by vasodilation, thereby increasing blood flow to under-oxygenated regions. In the pulmonary circulation of the lungs, however, the converse effect, hypoxic vasoconstriction, is critical for shunting blood to more highly ventilated regions to help optimize the ventilation of deoxygenated blood. Subunit 4 of cytochrome c oxidase (CcO) is a nuclear-encoded regulatory subunit of the terminal complex of the mitochondrial electron transport chain. We have recently discovered an isoform of CcO 4 (CcO4-2) which is specific to lung and trachea, and is induced after birth. The role of CcO as the major cellular oxygen consumer, and the lung-specific expression of CcO4-2, led us to investigate CcO4-2 gene regulation. We cloned the CcO4-2 promoter regions of cow, rat and mouse and compared them with the human promoter. Promoter activity is localized within a 118-bp proximal region of the human promoter and is stimulated by hypoxia, reaching a maximum (threefold) under 4% oxygen compared with normoxia. CcO4-2 oxygen responsiveness was assigned by mutagenesis to a novel promoter element (5¢-GGACGTTCCCACG-3¢) that lies within a 24-bp region that is 79% conserved in all four species. This element is able to bind protein, and competition experiments revealed that, within the element, the four core bases 5¢-TCNCA-3¢ are obligatory for transcription factor binding. CcO isolated from lung showed a 2.5-fold increased maximal turnover compared with liver CcO. We propose that CcO4-2 expression in highly oxygenated lung and trachea protects these tissues from oxidative damage by accelerating the last step in the electron transport chain, leading to a decrease in available electrons for free radical formation.Abbreviations CcO, cytochrome c oxidase; CcO4-2, CcO subunit 4-2 gene; Egr1, early growth response factor 1; EMSA, electrophoretic mobility shift assay; HIF-1a, hypoxia-inducible factor 1a; ORE, oxygen responsive element; OREF, ORE binding factor; RACE, rapid amplification of cDNA ends; ROS, reactive oxygen species.

Evolutionary GEM: Evolution of Cytochrome c Oxidase IV Regulation in Mammals

“…Early aerobes to optimize their metabolic profile in an O 2 -rich environment. This different pathway includes a novel oxygenresponsive element (ORE) comprising a 24-nucleotide sequence downstream of the previously described HRE within the COX4-2 promoter (8). Sequence conservation of the ORE between mammalian tissues suggests that the sequence may have been present in an ancestor of modern mammals (8).…”

“…This different pathway includes a novel oxygenresponsive element (ORE) comprising a 24-nucleotide sequence downstream of the previously described HRE within the COX4-2 promoter (8). Sequence conservation of the ORE between mammalian tissues suggests that the sequence may have been present in an ancestor of modern mammals (8). Furthermore, the ORE differs from the HRE with respect to its position and composition in the COX4-2 gene, thus illustrating its lack of homology (ancestral relationship) with the HIF1 pathway (8).…”

“…Sequence conservation of the ORE between mammalian tissues suggests that the sequence may have been present in an ancestor of modern mammals (8). Furthermore, the ORE differs from the HRE with respect to its position and composition in the COX4-2 gene, thus illustrating its lack of homology (ancestral relationship) with the HIF1 pathway (8). Furthermore, gene activity increased more than threefold when an ORE was present under hypoxic conditions, whereas HREs showed negligible activity under identical conditions (8).…”

“…Furthermore, the ORE differs from the HRE with respect to its position and composition in the COX4-2 gene, thus illustrating its lack of homology (ancestral relationship) with the HIF1 pathway (8). Furthermore, gene activity increased more than threefold when an ORE was present under hypoxic conditions, whereas HREs showed negligible activity under identical conditions (8). Conversely, when the ORE is mutated, COX4-2 gene activity plummets, thereby outlining its importance in the O 2 -dependent regulatory pathway of COX4-2 (8).…”

“…On the contrary, HIF1 and ORE mediated regulation may have converged over many generations to complement each other, ultimately maximizing cellular respiration during hypoxia. For instance, HIF1 is maximally expressed in complete absence of O 2, yet minimally expressed in hypoxia; the opposite holds true for ORE activity (8,9). A competitive advantage may have been gained by individuals who could account for slight, but potentially detrimental, changes in atmospheric O 2 .…”