| Description |
The Oct1/POU2F1 transcription factor was previously thought to constitutively occupy its cognate DNA binding sites, and to regulate the expression of housekeeping genes. This stereotype led to little attention being paid to Oct1 activity in dynamic cellular responses. In 2005, Oct1-/- mouse embryonic fibroblasts were shown to be highly sensitive to oxidative and genotoxic stresses, implicating Oct1 as a stress sensor. However, the mechanism connecting stress with Oct1 transcription regulation was unknown. To identify the mechanism by which Oct1 activity is regulated by posttranslational modifications in response to stress exposure, I used affinity purification and mass spectrometry to map specific Oct1 phosphorylation, O-GlcNAc modification and ubiquitination sites. Serine 385 (Chapter 2): I identified unique mechanisms of Oct1 regulation in response to stress and during mitosis, involving two different Oct1 phosphorylation events. Following genotoxic and oxidative stress, Oct1 binding specificity is mediated by phosphorylation of S385, switching from a monomeric into a dimeric conformation on different binding sequences. I confirmed this mechanism by genome wide ChIPseq. The homologous protein Oct4, a master regulator of embryonic stem cells, uses a similar mode of regulation. Serine 335 (Chapter 3): I identified an other phosphorylation event (pS335) as a negative regulator of Oct1 binding to DNA. I found that this phosphorylation is induced iv in mitotic cells as well as in stressed ones. The phosphorylated Oct1 is enriched in the mitotic spindle poles and midbody. Phospho-Oct1 is also K11-ubiquitinated. Using several different approaches, I showed that Oct1 directly regulates mitosis after being displaced from mitotic chromatin. Threonine 255 and Serine 728 (Chapter 4): In addition to phosphorylation and ubiquitination, I also identified O-GlcNAc modification of Oct1. Using an Oct1 glycosylation defective mutant, I found that the glycosylated residues of Oct1 regulate its DNA binding and transcriptional activity. Finally, I confirmed in embryonic stem cells that Oct1 and Oct4 share binding specificity for novel multimeric as well as conventional octamer motifs (Chapter 5). Further, multimeric binding motifs recruit Oct1 and Oct4 hetero-complexes suggesting extensive crosstalk between Oct1 and Oct4 in embryonic stem cells. |
