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X-inactivation normalizes O-GlcNAc transferase levels and generates an O-GlcNAc-depleted Barr body.

Olivier-Van Stichelen S, Hanover JA - Front Genet (2014)

Bottom Line: Given that OGT has an established role in polycomb repression, it is uniquely poised to auto-regulate its own expression through X-inactivation.In addition, we used chromatin isolation by RNA purification (ChIRP) and immunolocalization to examine O-GlcNAc levels in the Xi/Barr body.Despite the established role of O-GlcNAc in polycomb repression, OGT and target proteins bearing O-GlcNAc are largely depleted from the highly condensed Barr body.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health Bethesda, MD, USA.

ABSTRACT
O-GlcNAc Transferase (OGT) catalyzes protein O-GlcNAcylation, an abundant and dynamic nuclear and cytosolic modification linked to epigenetic regulation of gene expression. The steady-state levels of O-GlcNAc are influenced by extracellular glucose concentrations suggesting that O-GlcNAcylation may serve as a metabolic sensor. Intriguingly, human OGT is located on the X-chromosome (Xq13) close to the X-inactivation center (XIC), suggesting that OGT levels may be controlled by dosage compensation. In human female cells, dosage compensation is accomplished by X-inactivation. Long noncoding RNAs and polycomb repression act together to produce an inactive X chromosome, or Barr body. Given that OGT has an established role in polycomb repression, it is uniquely poised to auto-regulate its own expression through X-inactivation. In this study, we examined OGT expression in male, female and triple-X female human fibroblasts, which differ in the number of inactive X chromosomes (Xi). We demonstrate that OGT is subjected to random X-inactivation in normal female and triple X cells to regulate OGT RNA levels. In addition, we used chromatin isolation by RNA purification (ChIRP) and immunolocalization to examine O-GlcNAc levels in the Xi/Barr body. Despite the established role of O-GlcNAc in polycomb repression, OGT and target proteins bearing O-GlcNAc are largely depleted from the highly condensed Barr body. Thus, while O-GlcNAc is abundantly present elsewhere in the nucleus, its absence from the Barr body suggests that the transcriptional quiescence of the Xi does not require OGT or O-GlcNAc.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 2: 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.

Mentions: We next hypothesized that one allele of OGT is transcribed in each cell and that additional OGT alleles are associated with major components of the Barr body, such as XIST RNA and mH2A1. To determine the transcriptional status of OGT, we observed the localization of the OGT primary transcript in comparison to XIST lncRNA by RNA-FISH. The OGT primary transcript was detected using an intronic probe. We observed that XIST lncRNA spreads along the inactive X (Xi), and were able to observe 0, 1 or 2 spots in male, female and triple-X female (Figure 2A), respectively. In contrast, OGT primary transcript was only synthesized by the active X (Xa), and was synthesized by only one X-chromosome in male female or triple-X female (Figure 2A). As a positive control, PPIB [Bos Taurus Peptidylprolyl Isomerase B (cyclophilin B)] and RNA polymerase II mRNA were localized throughout the cells (Figure S1A). As a negative control, we were unable to detect bacterial DNA using 2 probes in these human cells (Figure S1A). This experiment confirms that OGT is synthesized only from one chromosome in these cell lines and that additional OGT alleles are silenced by the spread of XIST lncRNA.


X-inactivation normalizes O-GlcNAc transferase levels and generates an O-GlcNAc-depleted Barr body.

Olivier-Van Stichelen S, Hanover JA - Front Genet (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120696&req=5

Figure 2: 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.
Mentions: We next hypothesized that one allele of OGT is transcribed in each cell and that additional OGT alleles are associated with major components of the Barr body, such as XIST RNA and mH2A1. To determine the transcriptional status of OGT, we observed the localization of the OGT primary transcript in comparison to XIST lncRNA by RNA-FISH. The OGT primary transcript was detected using an intronic probe. We observed that XIST lncRNA spreads along the inactive X (Xi), and were able to observe 0, 1 or 2 spots in male, female and triple-X female (Figure 2A), respectively. In contrast, OGT primary transcript was only synthesized by the active X (Xa), and was synthesized by only one X-chromosome in male female or triple-X female (Figure 2A). As a positive control, PPIB [Bos Taurus Peptidylprolyl Isomerase B (cyclophilin B)] and RNA polymerase II mRNA were localized throughout the cells (Figure S1A). As a negative control, we were unable to detect bacterial DNA using 2 probes in these human cells (Figure S1A). This experiment confirms that OGT is synthesized only from one chromosome in these cell lines and that additional OGT alleles are silenced by the spread of XIST lncRNA.

Bottom Line: Given that OGT has an established role in polycomb repression, it is uniquely poised to auto-regulate its own expression through X-inactivation.In addition, we used chromatin isolation by RNA purification (ChIRP) and immunolocalization to examine O-GlcNAc levels in the Xi/Barr body.Despite the established role of O-GlcNAc in polycomb repression, OGT and target proteins bearing O-GlcNAc are largely depleted from the highly condensed Barr body.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health Bethesda, MD, USA.

ABSTRACT
O-GlcNAc Transferase (OGT) catalyzes protein O-GlcNAcylation, an abundant and dynamic nuclear and cytosolic modification linked to epigenetic regulation of gene expression. The steady-state levels of O-GlcNAc are influenced by extracellular glucose concentrations suggesting that O-GlcNAcylation may serve as a metabolic sensor. Intriguingly, human OGT is located on the X-chromosome (Xq13) close to the X-inactivation center (XIC), suggesting that OGT levels may be controlled by dosage compensation. In human female cells, dosage compensation is accomplished by X-inactivation. Long noncoding RNAs and polycomb repression act together to produce an inactive X chromosome, or Barr body. Given that OGT has an established role in polycomb repression, it is uniquely poised to auto-regulate its own expression through X-inactivation. In this study, we examined OGT expression in male, female and triple-X female human fibroblasts, which differ in the number of inactive X chromosomes (Xi). We demonstrate that OGT is subjected to random X-inactivation in normal female and triple X cells to regulate OGT RNA levels. In addition, we used chromatin isolation by RNA purification (ChIRP) and immunolocalization to examine O-GlcNAc levels in the Xi/Barr body. Despite the established role of O-GlcNAc in polycomb repression, OGT and target proteins bearing O-GlcNAc are largely depleted from the highly condensed Barr body. Thus, while O-GlcNAc is abundantly present elsewhere in the nucleus, its absence from the Barr body suggests that the transcriptional quiescence of the Xi does not require OGT or O-GlcNAc.

No MeSH data available.


Related in: MedlinePlus