Cellular context- and protein level-dependent interaction of pluripotency factor OCT4A with multiple octamer motifs of the same target gene

Aims: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. Materials and methods: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. Key findings: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. Significance: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.