LPS induces limited activation of hypoxia-inducible factor-1α in macrophages.

Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in cellular and tissue adaptation to low oxygen conditions. Although inflammatory stimuli such as lipopolysaccharide (LPS) also increase HIF-1α levels under normoxia, its transcriptional activity and regulatory mechanisms in this context remain unclear. To address this, we performed chromatin immunoprecipitation sequencing and transcriptome analyses in murine macrophages stimulated with either LPS or hypoxia. Both stimuli stabilized HIF-1α protein but via distinct mechanisms: hypoxia acted post-translationally, whereas LPS increased Hif-1α mRNA expression. Genome-wide HIF-1α binding was observed under both conditions; however, only hypoxia induced broad transcriptional activation of target genes, whereas LPS upregulated a restricted set, mostly glycolytic genes. Motif enrichment analysis revealed that hypoxia, but not LPS, promoted cooperative transcription factor engagement, including HIF-1β, ETS, and bZIP family members. Hypoxia also increased H3K27 acetylation at HIF-1α target loci, consistent with a transcriptionally permissive chromatin state. In contrast, LPS led to reduced H3K27ac at noninduced loci, suggesting epigenetic repression. Mechanistically, HIF-1α exhibited a phosphorylation-dependent band shift under hypoxia but not LPS. Although both conditions showed comparable overall phosphorylation levels by Phos-tag analysis, only hypoxia triggered a conformational change, suggesting site-specific phosphorylation linked to transcriptional competence. These findings demonstrate that HIF-1α binding alone is insufficient for gene activation and that phosphorylation and chromatin context determine its transcriptional output in a stimulus-dependent manner.