Genome-wide maps of H3K4Me3 histone modification in human pulmonary endothelial cells under hypoxia

Purpose: In lung tissue of mice with experimental PH, we identified an upregulated lncRNA 5031425E22 which mapped to a human ortholog KMT2E-AS1. Across mammals, this lncRNA gene sits adjacent (head-to-head) to the chromosomal location of the histone lysine N-methyltransferase 2E gene (KMT2E), a member of a family of regulators controlling histone 3 lysine 4 trimethylation (H3K4Me3) and chromatin remodeling. We found that both mouse lncRNA E22 and human KMT2E-AS1 were increased in multiple in vivo rodent and human instances of PH. This ChIP-sequencing study was designed to compare the histone 3 lysine 4 methylated regions/gene profile of human pulmonary arterial endothelial cells (HPAECs) under hypoxia as compared to normoxia. Method: Primary HPAECs were grown in EBM-2 basal medium supplemented with EGM-2 MV BulletKit (Lonza). Experiments were performed at passages 5 to 8. Cultured HPAECs were either under normoxia or hypoxia (0.2% oxygen) for 24 hours. Cells were fixed, crosslinked, and sonicated to get soluble chromatin for immunoprecipitation. 10% input of crosslinked DNA was saved while the remining DNA was precipitated with histone 3 lysine 4 antibody (H3K4Me3, Abcam, ab8580) or non-immune rabbit IgG (Pierce magnetic ChIP kit). Chromatin/antibody complex was then pulled down using Protein A/G Magnetic Beads (Pierece). DNA samples were proceeded with ChIP-sequencing (sequencing platform: DNBSeqTM, 20M reads). H3K4Me3 ChIP pulldown of HPAECs under hypoxia vs normoxia using 7 total samples: 1 control, 3 normoxia, and 3 hypoxia.