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

Abstract

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...

Establishment of a Human RNA Pol II Pausing System and the Identification of O-GlcNAc Cycling Regulating Pol II Pausing and Elongation

“…Previous experiments showed that inhibition of OGA in the CFS blocked elongation 14 , as effectively as the CDK9 and PARP-1 inhibitors in this study. Those experiments indicated that OGA catalytic activity was required for elongation and secondly, that OGT must have acted prior to that point in order to create a substrate for the OGA.…”

“…This is easily done by conjugating biotinylated-CMV promoter template to magnetic beads. Using this assay system, one can isolate various steps in the transcription processes wash off soluble factors, and then add back the activity in question to assess its function 14,30,31 . As indicated in Figure 3A, a 30" pulse was used to label RNA and transcription was stopped with EDTA.…”

“…In contrast to the serine/threonine kinases and phosphatases, there is only one transferase, the O-GlcNAc transferase (OGT) and one removal enzyme, O-GlcNAc aminidase (OGA) 35 . Previously it was shown that OGT is required for PIC formation 15,16 and that OGA is required for proper elongation in the CFS 14 . The OGA elongation defect directly implicates an OGT catalytic requirement, which must occur prior to elongation, since OGA activity requires an O-GlcNAcylated substrate.…”

O-GlcNAc Homeostasis Controls Cell Fate Decisions During Hematopoiesis

“…Regulation of transcription factors and co-activator/co-repressor complexes by O-GlcNAc is poorly understood. OGT is part of the mSin3A complex (Yang et al 2002) and the polycomb complex (Chu et al 2014;Maury et al 2015); and both OGA and OGT help organize the RNA Pol II pre-initiation complex (Ranuncolo et al 2012;Lewis et al 2016;Resto et al 2016). Recent advances in O-GlcNAc crosslinking technology will aid in the determination of which proteins interact with O-GlcNAcylated transcriptional complexes, how these complexes change in response to different stimuli, and potentially identify protein domains that preferentially bind to O-GlcNAcylated moieties (Rodriguez et al 2015).…”

“…OGT also interacts with mSin3A or components of the polycomb repressive complex (PRC) 1 and 2 to mediate transcription repression by forming stable repressor complexes (Yang et al 2002;Chu et al 2014;Maury et al 2015). OGT and OGA regulate preinitiation complex (PIC) formation and RNA Polymerase II (Pol II) activity (Ranuncolo et al 2012;Lewis et al 2016;Resto et al 2016).…”