Hepatocyte nuclear factor 1 alpha (HNF1A) regulates transcription of O‐GlcNAc transferase in a negative feedback mechanism

Abstract

O‐GlcNAc transferase (OGT)‐catalyzed protein O‐GlcNAcylation is implicated in diverse cellular events. In the present study, we report the regulation of ogt transcription by the hepatocyte nuclear factor 1 homologue A (HNF1A) in HEK293T cells. We first identified a core ogt promoter (−150 to +200 bp) and confirmed its binding to the transcription factor HNF1A. We found that HNF1A regulates ogt transcription in a time‐dependent manner and that O‐GlcNAcylation of HNF1A represses ogt transcription. Electron‐transfer dissociation based tandem mass spectrometry analysis revealed 14 O‐GlcNAc sites on HNF1A, six of which are predominantly modified, including Ser303/304, Ser471, Ser560 and Thr563/564. We further found that loss of O‐GlcNAcylation at Ser303/304 or Thr563/564 significantly elevates ogt transcription. These findings highlight a negative feedback mechanism for ogt transcription, which partially explains the homeostasis of cellular O‐GlcNAcylation.

Analytical and Biochemical Perspectives of Protein O-GlcNAcylation

“…OGT protein expression is well regulated. Besides tissue- and organelle-specific expression regulation, OGT expression undergoes transcriptional regulation upon stimuli (e.g., via activating/repressing OGT promoter activity). − Of special note, OGT expression can also be induced by altered expression of its counterpart OGA, suggesting that the two enzymes are coordinately regulated to maintain balanced O-GlcNAc levels for homeostasis. , Moreover, treatment of cells with the selective OGT inhibitors decreases O-GlcNAc levels but with a particularly large increase in OGT abundance and relatively smaller decrease in the OGA level observed (which might be ascribed by the compensatory increase in a correlated OGA gene expression) . OGT also undergoes post-transcriptional regulation, e.g., by alternative splicing and microRNAs (miRNAs). − Besides expression, OGT degradation is also tightly controlled (e.g., by ubiquitination). , …”

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

“…To test the fidelity of the conceptual dual fluorescent protein reporter, we compared its activity with that of a routine dual luciferase reporter assay. We have previously investigated ogt gene transcription in HEK293T cells using a routine method, which can report variant lengths of ogt promoters [ 24 ]. We assembled a long ogt promoter (−2000 to +200 bp) and a short promoter (−150 to +200 bp) before conducting assembly using the GFP reporter, and examined the reporter activity in HEK293T cells.…”

Glycaemia and body weight are regulated by sodium-glucose cotransporter 1 (SGLT1) expression via O-GlcNAcylation in the intestine

“…Our study showed that phosphorylation of CREB was markedly reduced in the Ogt -VKO mice, suggesting that O -GlcNAcylation might regulate Sglt1 transcription through the cAMP-CREB pathway. Other than CREB, we observed the expression of Sp-1 and HNF1 alpha, which were known to increase Sglt1 transcription [ 36 , 37 ] and to be O -GlcNAcylated [ 38 , 39 ]. Sp-1 and HNF1 alpha protein expression inversely increased in the Ogt -VKO mice.…”