O-GlcNAc and the Epigenetic Regulation of Gene Expression

Self Cite

We describe here the identification and functional characterization of the enzyme O-GlcNAcase (OGA) as an RNA polymerase II elongation factor. Using in vitro transcription elongation assays, we show that OGA activity is required for elongation in a crude nuclear extract system, whereas in a purified system devoid of OGA the addition of rOGA inhibited elongation. Furthermore, OGA is physically associated with the known RNA polymerase II (pol II) pausing/elongation factors SPT5 and TRIM28-KAP1-TIF1␤, and a purified OGA-SPT5-TIF1␤ complex has elongation properties. Lastly, ChIP-seq experiments show that OGA maps to the transcriptional start site/5 ends of genes, showing considerable overlap with RNA pol II, SPT5, TRIM28-KAP1-TIF1␤, and O-GlcNAc itself. These data all point to OGA as a component of the RNA pol II elongation machinery regulating elongation genome-wide. Our results add a novel and unexpected dimension to the regulation of elongation by the insertion of O-GlcNAc cycling into the pol II elongation regulatory dynamics.RNA polymerase II is the species of polymerase that transcribes the protein-coding genes in the cell. Largely based on in vitro transcription data and bacterial transcriptional regulation, it was thought that the pathway of transcription initiation was the de novo assembly and recruitment of general factors and pol II 4 into a preinitiation complex at promoters. In other words, transcription was solely controlled by whether initiation occurred or not. However, in the late 1980s, a transcriptionally engaged pol II was found on several genes, located roughly at ϩ50 relative to the transcriptional start site (TSS) (1-3). More recently, genome-wide approaches have shown that paused pol II exists on at least 40% of promoters in the genome (4 -9).These data show that the regulation of elongation is a significant and widespread method by which cells regulate gene expression.Biochemically, the establishment of a paused polymerase requires the recruitment of DSIF and NELF to pol II early in elongation to prevent pol II from productive elongation (10 -13). Release of the paused polymerase is achieved with P-TEFb phosphorylation of DSIF and NELF, ejecting NELF from pol II and converting DSIF into a positive elongation factor (14,15). The more recent discoveries of additional factors likely involved in pause establishment and release include human capping enzyme (16), the TFIIH-associated kinase, CDK7 (17, 18), the TFIIH ERCC3 helicase (19), , Integrator (23,24), ELL (25), TFIIS (26), TRIM28-KAP1-TIF1␤ (27, 28), Top1 (29), SEC (30), PAF complex (31, 32), and Gdown1 (33). The plethora of factors suggests that more complicated dynamics are at play in regulating pausing and elongation. Additionally, it is not clear, and indeed difficult to know, whether all of the factors involved in pol II pausing and elongation have been identified. This difficulty is due mostly to the absence of a human cell-based in vitro transcription system that recapitulates a paused polymerase and with which the full complement of...

mentioning

confidence: 99%

O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β

Resto

Kim

Fernandez

et al. 2016

Self Cite

“…O-GlcNAcylation plays an important role in controlling transcription. O-GlcNAc regulates gene transcription by directly modifying transcription factors/cofactors or RNA Pol II CTD (33) or by altering how the activity of these factors is modulated by other post-translational modifications (39). A previous study by Ranuncolo et al (33) suggested that an O-GlcNAcylation cycle on RNA Pol II CTD occurs at gene promoters.…”

Section: Discussionmentioning

confidence: 99%

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter

Zhang

Costa²,

Tan

et al. 2016

One mode of ␥-globin gene silencing involves a GATA-1⅐FOG-1⅐Mi2␤ repressor complex that binds to the ؊566 GATA site relative to the A ␥-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the ؊566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the A ␥-globin promoter at the ؊566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ␤-globin locus yeast artificial chromosome (␤-YAC) bone marrow cells. When WT ␤-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2␤ at the A ␥-globin promoter is increased. In addition, OGT and Mi2␤ recruitment is increased at the A ␥-globin promoter when ␥-globin becomes repressed in postconception day E18 human ␤-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2␤ is modified with O-GlcNAc, and both OGT and OGA interact with Mi2␤, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of ␥-globin gene regulation mediated by modulating the assembly of the GATA-1⅐FOG-1⅐Mi2␤ repressor complex at the ؊566 GATA motif within the promoter.

“…The role of O-GlcNAc in epigenetics is reviewed in detail in Ref. 98, whereas a few specific examples potentially relevant to cancer will be noted here.…”

Section: O-glcnac and Cancer Cell Epigeneticsmentioning

confidence: 99%

Cancer Metabolism and Elevated O-GlcNAc in Oncogenic Signaling

Vosseller

2014

184

145

O-Linked ␤-N-acetylglucosamine (O-GlcNAc) is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. Analogous to phosphorylation, O-GlcNAcylation plays crucial regulatory roles in cellular signaling. Recent work indicates that increased O-GlcNAcylation is a general feature of cancer and contributes to transformed phenotypes. In this minireview, we discuss how hyper-O-GlcNAcylation may be linked to various hallmarks of cancer, including cancer cell proliferation, survival, invasion, and metastasis; energy metabolism; and epigenetics. We also discuss potential therapeutic modulation of O-GlcNAc levels in cancer treatment.