Regulation of Oct1/Pou2f1 transcription activity by O ‐GlcNAcylation

Abstract

The Oct1 transcription factor is a potent regulator of stress responses, metabolism, and tumorigenicity. Although Oct1 is regulated by phosphorylation and ubiquitination, the presence and importance of other modifications is unknown. Here we show that Oct1 is modified by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. We map two sites of O-GlcNAcylation at positions T255 and S728 within human Oct1. Under anchorage-independent overgrowth conditions, Oct1 associates 3-fold more strongly with the Gadd45a promoter and mediates transcriptional repression. Increased binding correlates with quantitative reductions in Oct1 nuclear periphery-associated puncta, and a reduced association with lamin B1. The O-GlcNAc modification sites are important for both Gadd45a repression and anchorage-independent survival. In contrast to chronic overgrowth conditions, following acute nutrient starvation Oct1 mediates Gadd45a activation. The O-GlcNAc sites are also important for Gadd45a activation under these conditions. We also, for the first time, identify specific Oct1 ubiquitination sites. The findings suggest that Oct1 integrates metabolic and stress signals via O-GlcNAc modification to regulate target gene activity.

The Oct1 transcription factor and epithelial malignancies: Old protein learns new tricks

“…The opposite result – a more glycolytic phenotype – was observed in cells overexpressing Oct1. Wild-type immortalized MEFs (3T3 cells) die after 5–6 days in culture medium lacking glucose, whereas Oct1 deficient MEFs arrest but remain viable [44, 46, 52]. Consistent with these changes, multiple Oct1 targets, including stress- and signal-responsive target genes, encode metabolic regulators [46, 47, 53, 54].…”

“…Other Oct1/POU2F1 phosphorylation events have been documented in large screens but are not functionally annotated [58, 59]. Mass spectroscopy indicates that the protein is both ubiquitylated and O -GlcNAcylated [52]. The ubiquitin ligase(s) and deubiquitinases are unknown, as is the significance of the ubiquitylation events, but it is known that Oct1 is stabilized at the protein level in stem cells (see below).…”

Oct transcription factors in development and stem cells: insights and mechanisms

“…Oct1 can similarly reversibly associate with the nuclear periphery through interactions with lamin B1. In this latter case association is known to be regulated by oxidative stress, chronic overgrowth and cellular aging (Imai et al, 1997;Malhas et al, 2009;Kang et al, 2013).…”

“…These modifications are likely to account for many of the changes in levels, activity and localization described above. The two best-studied Oct proteins in this respect are again Oct4 and Oct1, which are regulated by phosphorylation (Segil et al, 1991;Nieto et al, 2007;Schild-Poulter et al, 2007;Kang et al, 2009;Van Hoof et al, 2009;Kang et al, 2011;Brumbaugh et al, 2012;Lin et al, 2012), O-GlcNAcylation (Webster et al, 2009;Jang et al, 2012;Kang et al, 2013), SUMOylation (Wei et al, 2007;Zhang et al, 2007) and ubiquitylation (Xu et al, 2004;Kang et al, 2011;Kang et al, 2013). These modifications control protein stability, their DNA binding properties, co-factor association and tethering to the nuclear envelope.…”

“…Oct1 has been shown to associate not only with transcription factors such as Sox2, C/EBPβ, AP-1 (Fos), NFAT, NFκB and Jang et al, 2012;Kang et al, 2013 glucocorticoid receptor (Li-Weber et al, 1998;Préfontaine et al, 1999;Belsham and Mellon, 2000;Hatada et al, 2000;van Heel et al, 2002), but also with itself and with other Oct proteins such as Oct2 and Oct6 to form homodimeric, heterodimeric and higherorder complexes (Verrijzer et al, 1992;Reményi et al, 2001). Interestingly, the ability to form these complexes can be controlled by phosphorylation of Oct proteins at specific sites in the DNAbinding domain (Nieto et al, 2007;Kang et al, 2009).…”