O -linkage of N -acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability

Abstract

, an enzyme recently shown to be necessary for cell survival (2). UDP-GlcNAc, which is synthesized de novo from glucose via the hexosamine biosynthetic pathway serves as the substrate for protein O-GlcNAcylation. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins (3, 4) as well as with transcriptional levels of some genes (5-9). However, how glucose flux through the hexosamine pathway regulates gene transcription remains elusive. Based on the observation that many transcription factors are modified by O-GlcNAc (10, 11), most often in the transcriptional activation domain (10, 11), the intriguing hypothesis has been raised that O-GlcNAcylation of transcription factors could regulate gene transcription in response to glucose flux (8-10, 12). Because O-GlcNAcylation and phosphorylation often occur reciprocally in transcription factors (1, 9, 13), it has been difficult to elucidate the direct effects of O-GlcNAc on transcriptional regulation. Our finding that a region in the activation domain of the transcription factor Sp1 is exclusively subject to O-GlcNAcylation, but not to phosphorylation, allows us to assess the functional roles of O-GlcNAc in transcription directly (12).The first transcription factor shown to bear the O-GlcNAc modification was Sp1 (10), a ubiquitous transcription factor that plays a vital role in the control of TATA-less housekeeping gene transcription (14). The N-terminal portion of the molecule contains two glutamine-rich activation domains, each associated with a serine-͞threonine-rich region, whereas the C-terminal region contains the zinc-finger DNA-binding domain (15). The second glutamine-rich activation domain has been shown to be involved both in the homomultimerization of Sp1 (16) and in the interaction with the general transcription factor (TF II D) via TATA-binding protein-associated factor II 110 (TAF II 110) (17, 18). The homomultimerization is required for synergistic activation of transcription by Sp1 (19) whereas the interaction with TF II D via TAF II 110 is required for the effect of Sp1 on DNA polymerase II-dependent transcription. These protein-protein interactions appear to be dependent on the glutamine͞ hydrophobic patches within this domain of Sp1 (18), a motif that is conserved in the activation domains of other transcription factors such as CREB and VP16 (20,21). Furthermore, we have identified a dominant O-GlcNAcylation site in the C-terminal region of this domain (12). Based on these characteristics of this glutamine-rich activation domain of Sp1, we developed a model Sp1 peptide, termed SpE, which spans this domain and in which we could control the O-GlcNAc state either by mutagenesis or by use of different expression systems. Using an in vitro pulldown assay, we showed that the interaction of SpE with either the full-length Sp1 or TAF II 110 was inhibited dramatically by O-GlcNAcylation of the SpE peptide, suggesting that the OGlcNAc residue interrupted the hydrophobic interactions between the SpE peptide and its partners...

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

“…As described in several reviews 2,4,35 , O-GlcNAc glycosylation has been shown to both enhance and suppress the activity of transcription factors. O-GlcNAc can disrupt protein-protein interactions, as in the case of Sp1, whose glycosylation represses transcription at Sp1-driven promoters 36,37 . In other cases, it can promote protein-protein interactions, as in the case of signal transducer and activator of transcription 5A (STAT5A), whose glycosylation enhances its activity by recruiting the transcriptional coactivator CREB-binding protein (CBP) 38 .…”

Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia

“…Sp1 was the first of a large number of Pol II transcription factors shown to carry the O-GlcNAc modification (35). Addition of O-GlcNAc has been shown to affect rates of proteasomal degradation and transcriptional activity of Sp1 (15,42). The effects of glucose on regulation of plasminogen activator inhibitor 1 and transforming growth factor-␤, proteins that are thought to play a role in the complications of diabetes, have also been shown to correlate with the degree of Sp1 glycosylation (43).…”

“…There was no relationship between GLUT4-OGT copy number and OGT expression or transgenic phenotype. On the basis of the Southern analysis and expression levels, five founder mice (15,18,23,26, and 32) were chosen to breed into a C57BL6 background. Presence of the OGT transgene mRNA in muscle (Fig.…”

“…A leading hypothesis suggests that the terminal metabolite of the pathway, UDP-GlcNAc, is used as a substrate by the recently cloned O-linked GlcNAc transferase (OGT) (11)(12)(13)(14). O-linked glycosylation by GlcNAc modifies the serine and threonine residues of cytosolic and nuclear proteins and, like phosphorylation, can change the function of such proteins as Sp1 and endothelial nitrogen oxide synthase (15,16). Given the relationship between the hexosamine pathway and the development of insulin resistance, we sought to determine whether these effects were mediated through the O-glycosylation pathway.…”

Interplay between HIV-1 Vpr and Sp1 Modulates p21WAF1 Gene Expression in Human Astrocytes

“…The DNA binding and transcriptional activities of Sp1 are regulated by the state of phosphorylation that follows changes in the cell cycle (24). Sp1 protein is stabilized by O-linked glycosylation, which confers resistance to proteasome-dependent degradation (19,25).…”