An optimized reporter of the transcription factor hypoxia-inducible factor 1α reveals complex HIF-1α activation dynamics in single cells

Abstract

Immune cells adopt a variety of metabolic states to support their many biological functions, which include fighting pathogens, removing tissue debris, and tissue remodeling. One of the key mediators of these metabolic changes is the transcription factor hypoxia-inducible factor 1α (HIF-1α). Single-cell dynamics have been shown to be an important determinant of cell behavior; however, despite the importance of HIF-1α, little is known about its single-cell dynamics or their effect on metabolism. To address this knowledge gap, here we optimized a HIF-1α fluorescent reporter and applied it to study single-cell dynamics. First, we showed that single cells are likely able to differentiate multiple levels of prolyl hydroxylase inhibition, a marker of metabolic change, via HIF-1α activity. We then applied a physiological stimulus known to trigger metabolic change, interferon-γ, and observed heterogeneous, oscillatory HIF-1α responses in single cells. Finally, we input these dynamics into a mathematical model of HIF-1α-regulated metabolism and discovered a profound difference between cells exhibiting high versus low HIF-1α activation. Specifically, we found cells with high HIF-1α activation are able to meaningfully reduce flux through the tricarboxylic acid cycle and show a notable increase in the NAD⁺/NADH ratio compared with cells displaying low HIF-1α activation. Altogether, this work demonstrates an optimized reporter for studying HIF-1α in single cells and reveals previously unknown principles of HIF-1α activation.

Keywords: fluorescence; hypoxia-inducible factor (HIF); mathematical modeling; microscopy; systems biology.