Fig 2: Analysis of FASN, OGT and O-GlcNAcylation contents in human hepatic cell lines. (A) Expression of FASN, OGT and O-GlcNAcylation was evaluated by western blot in three different hepatic cell lines, the liver cancer-derived cell lines HepG2 and Hep3B and the immortalized human hepatocytes IHH. (B) Quantification of three independent experiments from (A). *P<0.05. NS, non-significant; FASN, fatty acid synthase; OGT, O-GlcNAc transferase; IHH, immortalized human hepatocyte.

Fig 3: OGT is expressed from Xa and extra-OGT alleles are part of the X-inactivation complex. (A) RNA-FISH analysis of the OGT primary transcript and XIST lncRNA in male, female and triple-X female cells indicates that OGT expression does not co-localize with the Xist lncRNA. Xa: active X-chromosome; Xi: inactive X-chromosome. (B) Chromatin isolation by RNA Purification (ChIRP) of XIST RNA, a major component of Barr body, shows interaction of the OGT locus with XIST mRNA. As a control, G6PD, another X-linked gene is also found to interact, but not SRY, which is on the Y chromosome. (C) Chromatin-Immunoprecipitation of mH2A1, another key component of the Barr body demonstrates interactions with the OGT gene and XIST mRNA. (D) Chromatin-Immunoprecipitation of mH2A1 also demonstrates that no O-GlcNAcylated proteins are associated with the immuno-precipitated complexes.

Fig 4: Barr bodies do not require O-GlcNAc cycling. (A) A strong-DAPI staining signal and/or a lack of elongating RNA polymerase II (PhosphoS2) signal were used to localize Barr bodies (white arrows). O-GlcNAc staining is largely excluded from Barr body. (B) OGT is also less intense in Barr body. (C) Unlike elongating RNA polymerase II (PhosphoS2), unmodified RNA polymerase II co-localized with the Barr body. (D) Barr bodies were identified as in this figure. H3K27me3 strongly co-localized with Barr bodies, suggesting a highly silenced region. (E) mH2A1 also co-localized with Barr bodies. In both experiments, O-GlcNAc staining did not co-localize with these Barr body-specific staining regions. (F) RNA-FISH analysis of the OGT primary transcript and XIST lncRNA in female and triple-X female, in presence of azaserine of Thiamet G. Xa: active X-chromosome; Xi: inactive X-chromosome. Immunofluorescence pictures are representative of triplicate experiments.

Fig 5: OGT expression is regulated by X-inactivation in female mammals. The X-inactivation process begins with a counting/choice of which chromosome to inactivate. X-inactivation leads to silencing of almost the entire X-chromosome, including the XIST-proximal OGT gene, by recruitment of polycomb group proteins via XIST lncRNA and appearance of DNA repressive marks such as H3K27me3, H2AK119Ub and mH2A1. Once inactivation is completed, the inactivated X-chromosome (Xi) is re-localized in a highly compacted structure called the Barr body, from which OGT protein and O-GlcNAcylation are largely excluded indicating a highly transcriptionally silenced region. This process allows the dosage of OGT protein to be normalized in expression independent of the number of X-chromosomes. Escape from X-inactivation can lead to increases in OGT expression. Xa: active X-chromosome.