O-GlcNAcylation of human TATA-Box binding protein is required to sustain key metabolic enzyme gene expression

Cellular homeostasis is ensured by myriad cellular processes that integrate environmental changes and maintain stability within the organism. Variation in nutrient availability can be reflected by the post-translational modification of many proteins by the nutrient sensor O-GlcNAcylation. Herein, we describe a molecular mechanism of transcription regulation by O-GlcNAcylation of the TATA-box binding protein (TBP). We show that O-GlcNAcylation regulates its interaction with BTAF1, hence, formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. We mapped three O-GlcNAcylation sites at the N-terminus of TBP and defined T114 as a main regulator of the interaction with BTAF1. CRISPR/Cas9 editing of wild-type TBP replaced by a T114A mutant, leads to profound modification of HeLa cells' glucose and lipid metabolism and gene expression profile. These data indicate that basal transcription machinery, via O-GlcNAcylation of TBP, can integrate nutrient availability and modulate the transcriptome, resulting in adaptation of cellular metabolism. Overall design: Endogenous TBP gene was edited using CRISPR/Cas9 technology in HeLa cells. The 3X-FLAG was inserted in phase at the N-terminal of the protein and the T114 O-GlcNAcylation site was replaced by an alanine. RNAs of the parental HeLa cell line as well as HeLa TBP3X-WT and HeLa TBP3X-T114A cell lines were extracted using RNeasy plus kit (QIAGEN) following manufacturer's instructions. Samples from each cell lines were submitted in triplicate to total RNA deep sequencing. Library preparation, sequencing and data analysis were performed at the Johns Hopkins University Deep Sequencing and Microarray Core Facility. RNA-Seq libraries were constructed using TruSeq stranded Total mRNA library kit (Illumina, Inc.) and 75 cycle runs were performed in multiplex on the NextSeq 500 platform.