Human PARP1 Knockout Cell Line-HEK293T

In human melanoma, PARP-1 and HIF-1α expression are tightly linked. Upon hypoxic stimulation, poly(ADP-ribose) (PAR) is synthesized and HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation of specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach, researchers demonstrate that PARP-1 dictates hypoxia-dependent HIF recruitment to chromatin at a range of HIF-regulated genes, while analysis of HIF-binding motifs (RCGTG) reveals that recognition of hypoxia-responsive elements is restricted in the absence of PARP-1. As a result, cells are less adaptable to hypoxia and exhibit reduced fitness during hypoxia induction. These data describe a fine-tuned regulation of HIF activation by PARP-1/PARylation and suggest that PARP inhibitors may have therapeutic potential for cancer types that exhibit HIF-1α hyperactivation.

ChIP-Seq analysis was performed on HEK 293T WT and HEK 293T PARP-1 knockout (PARP-1 KO) cells during early hypoxia (4 hours). ChIP-Seq showed that the majority of HIF-1α binding peaks detected were located within the first kilobase upstream of the translation start site: 48.16% for wild type and 43.9% for PARP-1 KO (Figure 1), which includes the 5′-UTR and promoter region of the gene. Statistical analysis was completed to eliminate non-significant peaks and compare them to input and normoxic control signals. 123 peaks were detected in hypoxic wild type cells and 68 peaks were detected in PARP-1 KO cells.

Figure 1. Chip-seq analysis of HIF-1α capacity to bind the promoters of its target genes on WT vs. PARP-1 knockout cells. (Martí, Juan Manuel, et al. 2021)