Whelan, S. A. | Search bioRxiv

bioRxiv2024

Multi-Omics after O-GlcNAc Alteration Identifies Cellular Processes Working Synergistically to Promote Aneuploidy

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can synergistically fine-tune multiple cellular pathways.

cell biology↗

bioRxiv2021

Regulation of Liver Regeneration by hepatocyte O-GlcNAcylation in mice

The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here we report that O-GlcNAcylation, an intracellular post-translational modification (PTM) regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration (LR) following partial hepatectomy (PHX). We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively. OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-Seq studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and IHC analysis. Furthermore, a significant decrease in HNF target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4 is O-GlcNAcylated in normal differentiated hepatocytes. These studies show that O-GlcNAcylation plays a critical role in the termination of LR via regulation of HNF4 in hepatocytes. Layman summaryO-GlcNAcylation is a protein modification that plays a critical role in various biological processes including cell proliferation, differentiation, and disease progression. These studies show that O-GlcNAcylation in hepatocytes is essential for proper liver regeneration. Without O-GlcNAcylation, hepatocytes keep on proliferating eventually forming liver tumors.

pathology↗