Hypoxia-reoxygenation alters global and gene-specific DNA methylation of GAPDH, independently of its expression and enzyme activity in the shrimp Penaeus vannamei.

Hypoxia promotes a shift from aerobic to anaerobic metabolism. In the shrimp Penaeus vannamei, low oxygen modulates glycolytic enzymes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Because hypoxia can also trigger epigenetic modifications, GAPDH could be a useful model to examine oxygen-sensing epigenetic interactions in shrimp. The GAPDH promoter was characterized, and global and gene-specific DNA methylation, DNA oxidation (8-OHdG), gene expression, enzymatic activity, and glucose/lactate during hypoxia-reoxygenation were measured. Three 1 kb fragments upstream of the GAPDH gene were obtained and sequenced. The presence of CpG islands, putative HIF-1α, p53, and TBP binding sites was analyzed in silico. Comparisons of the GenBank genome annotation with cross-species protein alignments and AlphaFold3 modeling suggest that the P. vannamei GAPDH gene may have an anomalous extension. Global DNA methylation increased under hypoxia at 24 h (P < 0.05) and peaked during reoxygenation at 12 h (P < 0.05). 8-OHdG peaked during hypoxia at 6-12 h (P < 0.05) and declined during reoxygenation. Promoter region methylation (6.9-10%) remained stable despite changes in expression, whereas intragenic methylation (1% in the control) increased under hypoxia and early reoxygenation (P < 0.05) without consistently following transcript levels. GAPDH activity increased under hypoxia with a 12 h peak (P < 0.05), returned to baseline at 24 h, and increased during reoxygenation (P < 0.05). Glucose and lactate changed inversely under hypoxia, but varied in reoxygenation. Overall, GAPDH appears to be modulated by oxygen-sensitive transcriptional or metabolic control rather than promoter methylation; intragenic methylation, global methylation, and 8-OHdG reflected stress.