Limits...
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

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

License
getmorefigures.php?uid=PMC4120696&req=5

Figure 3: 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.

Mentions: Previous studies have shown that O-GlcNAc is an abundant modification in interphase nuclei, and found highly concentrated at nuclear pores and subdomains within the nucleus (Hanover, 2001). Similarly, we detected an intranuclear distribution of OGT (Figure 3). We sought to determine whether O-GlcNAc might be associated with Barr bodies. The Barr body is known to have a unique chromatin signature maintained by excluding some chromatin modifiers while enriching others (Chadwick and Willard, 2003). Morphological inspection of the Barr body in interphase nuclei has proven to be a useful tool in order to look at numerous chromatin modifications including methylation and acetylation (Chadwick and Willard, 2003). To identify the Barr body, we initially looked for the exclusion of elongating RNA polymerase II (phospho-S2) staining on the periphery of the nucleus. With immunostaining of elongating RNA polymerase II, we identified 0, 1 or 2 Barr bodies in male (XY), female (XX) and triple-X (XXX) human fibroblasts, respectively (Figures 3A,B; white arrows). The identified Barr bodies were also DAPI-rich as it has been reported (Peters et al., 2002), likely due to an increased amount of chromatin compaction of heterochromatin. Surprisingly, the Barr bodies lacked staining of O-GlcNAcylation (Figure 3A) and OGT (Figure 3B). In order to demonstrate that the exclusion of O-GlcNAcylation was not due to an artifact of a specific antibody, we confirmed the exclusion using another phospho-S RNA polymerase II antibody (phospho S2/5) (Figure S1B). Single secondary antibody staining also confirmed the specificity of observed signals (Figure S1C). However, unphosphorylated RNA polymerase II colocalized with the Barr bodies (Figure 3C), which were identified in this set of experiments by both a lack of O-GlcNAc staining and strong DAPI staining. Together, these data show that the Barr bodies exclude only the elongating form of RNA polymerase II. Exclusion of O-GlcNAc from the inactive X was confirmed by comparing O-GlcNAc staining with H3K27Me3 and mH2A1 staining, both highly enriched in the Barr body (Figures 3E,F).


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

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

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

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

Figure 3: 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.
Mentions: Previous studies have shown that O-GlcNAc is an abundant modification in interphase nuclei, and found highly concentrated at nuclear pores and subdomains within the nucleus (Hanover, 2001). Similarly, we detected an intranuclear distribution of OGT (Figure 3). We sought to determine whether O-GlcNAc might be associated with Barr bodies. The Barr body is known to have a unique chromatin signature maintained by excluding some chromatin modifiers while enriching others (Chadwick and Willard, 2003). Morphological inspection of the Barr body in interphase nuclei has proven to be a useful tool in order to look at numerous chromatin modifications including methylation and acetylation (Chadwick and Willard, 2003). To identify the Barr body, we initially looked for the exclusion of elongating RNA polymerase II (phospho-S2) staining on the periphery of the nucleus. With immunostaining of elongating RNA polymerase II, we identified 0, 1 or 2 Barr bodies in male (XY), female (XX) and triple-X (XXX) human fibroblasts, respectively (Figures 3A,B; white arrows). The identified Barr bodies were also DAPI-rich as it has been reported (Peters et al., 2002), likely due to an increased amount of chromatin compaction of heterochromatin. Surprisingly, the Barr bodies lacked staining of O-GlcNAcylation (Figure 3A) and OGT (Figure 3B). In order to demonstrate that the exclusion of O-GlcNAcylation was not due to an artifact of a specific antibody, we confirmed the exclusion using another phospho-S RNA polymerase II antibody (phospho S2/5) (Figure S1B). Single secondary antibody staining also confirmed the specificity of observed signals (Figure S1C). However, unphosphorylated RNA polymerase II colocalized with the Barr bodies (Figure 3C), which were identified in this set of experiments by both a lack of O-GlcNAc staining and strong DAPI staining. Together, these data show that the Barr bodies exclude only the elongating form of RNA polymerase II. Exclusion of O-GlcNAc from the inactive X was confirmed by comparing O-GlcNAc staining with H3K27Me3 and mH2A1 staining, both highly enriched in the Barr body (Figures 3E,F).

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