Journal of Molecular Cell Biology Advance Access originally published online on August 11, 2009
Journal of Molecular Cell Biology 2009 1(1):11-12; doi:10.1093/jmcb/mjp008
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A Little Bit of Sugar Makes Polycomb Better
Department of Molecular Biology and Biochemistry, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA
* Correspondence to: Yuri B. Schwartz, Tel: +1 732 445 6896, E-mail: schwartz{at}biology.rutgers.edu; Vincenzo Pirrotta, Tel: +1 732 444 2446, E-mail: pirrotta{at}biology.rutgers.edu
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Transcriptional repression by Polycomb group (PcG) proteins is now sweetened by the discovery of the essential role of O-GlcNAc glycosylation in the process. PcG protein polyhomeotic may be the key target, but alternative or additional functions including repression of transcription through glycosylation of the C-terminal domain of RNA polymerase II are also possible.
Polycomb group (PcG) proteins are transcriptional repressors intimately involved in the control of differential expression of the genome throughout development of nearly all multicellular eukaryotes. PcG proteins function as parts of large multiprotein complexes three of which, PRC1, PRC2 and PhoRC, have been characterized and shown to cooperate in the repression of a target gene. In Drosophila melanogaster, the model organism in which PcG mechanisms are most thoroughly studied, the repression of target genes is effected through discrete DNA elements called Polycomb response elements (PREs). PcG complexes are recruited to PREs via interactions with multiple sequence specific DNA binding proteins (for more comprehensive review see Schwartz and Pirrotta, 2007).
As the differentiation program unfolds, some of the PcG targets need to change state from repressed to active and vice versa, implying a dynamic regulation of PcG mechanisms. Post-translational modification of PcG proteins is likely to be involved in the process. Much is still to be learned about the kinds and roles of post-translational modifications that target PcG proteins but we know that phosphorylation of MEL18 and BMI1, both components of the human PRC1 complex and of EZH2, the histone methyltransferase component of PRC2, affects their association with chromatin or with their corresponding protein complex and affects their function (Elderkin et al., 2007; Schwartz and Pirrotta, 2007 and references therein).
Now Gambetta et al. (2009) add protein glycosylation to the repertoire of post-translational modifications involved in regulation of PcG repression. In a recent Science paper, Gambetta et al. (2009) show that the Drosophila PcG gene super sex combs (sxc) encodes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), the evolutionarily conserved enzyme which attaches O-linked β-N-acetylglucosamine (O-GlcNAc) at serine and threonine residues on a wide variety of nuclear and cytoplasmic proteins (Hart et al., 2007). The sxc/Ogt mutants that lack OGT expression or express truncated proteins with no catalytic activity lose all cellular O-GlcNAcylation and show severe derepression of multiple PcG target genes, strongly suggesting that O-GlcNAcylation is essential for PcG repression. Surprisingly, when Gambetta et al. mapped the genome-wide localization of chromosomal O-GlcNAcylated proteins, they found that the most enriched sites corresponded to some of the known or presumptive PREs. This striking observation prompted the authors to test whether any of the PcG proteins themselves are O-GlcNAcylated. Using wheat germ agglutinin (WGA) agarose as affinity matrix for purification of O-GlcNAcylated proteins, Gambetta et al. indeed identified Ph, but not any other of the tested components of PRC1, PRC2 or PhoRC, as a target of glycosylation. O-GlcNAcylation of Ph appears to be evolutionarily conserved as a recent mass spectrometric study has identified PHL3, one of the murine orthologs of Ph, as a target of O-GlcNAcylation (Chalkley et al., 2009). The authors speculate that glycosylation of Ph may be the function of Sxc/Ogt in PcG silencing. However, the requirement for Ph O-GlcNAcylation for PcG repression remains unclear. Chromatin immunoprecipitation experiments indicate that binding of PRC1, PRC2 or PhoRC1 components in sxc/Ogt mutants is largely unaffected, suggesting that the function of glycosylation is downstream of the recruitment of PcG complexes to PREs.
The unexpected intimate connection between O-GlcNAcylation and PcG repression is now beyond doubt yet several critical questions beg further investigation. It remains unclear whether the presence of O-GlcNAcylated proteins is inherent to all PREs or is specific only to some. It is also not obvious whether the high enrichment of O-GlcNAcylated proteins at PREs reflects the binding of glycosylated Ph or is caused by the presence of some other glycosylated protein(s). We note that two decades ago Jackson and Tjian (1988) reported that several isoforms of GAGA associated factor, a DNA-binding protein involved in recruitment of PcG complexes to many PREs, are O-GlcNAcylated.
Perhaps more important is the question of whether SXC/OGT itself is a chromosomal protein and binds to PREs. No experiment directly addressing this question has been done but the reported nuclear localization of murine OGT (Yang et al., 2008) makes it a viable possibility. Potential binding of SXC/OGT to PREs opens up the exciting possibility that O-GlcNAc glycosylation may be, in fact, the functional effector of the transcriptional repression itself. Intriguingly, the C-terminal domain (CTD) of calf thymus RNA polymerase II (Pol II) has been found to be heavily O-GlcNAcylated (Kelly et al., 1993) and glycosylation of CTD appears to be mutually exclusive and inhibitory for its phosphorylation (Comer and Hart, 2001). The wealth of experimental data (reviewed in Phatnani and Greenleaf, 2006) indicates that CTD phosphorylation is essential for the transition from transcription initiation to elongation. It is thus tempting to envision the O-GlcNAcylation of CTD by PRE-bound SXC/OGT as the activity directly inhibiting transcription. We should note that Gambetta et al. did not detect any glycosylated Pol II in Drosophila cells. This, however, may be explained by the fact that glycosylated Pol II binds poorly to WGA immobilized on agarose beads (Kelly et al., 1993), the affinity matrix used in the experiment of Gambetta et al. Clearly more work is needed to dissect the mechanistic link between O-GlcNAc glycosylation and PcG repression. Until then, we should keep in mind other possibilities: a large number of nuclear and cytoplasmic proteins are known to be O-GlcNAcylated (Hart et al., 2007) and the role of SXC/OGT in PcG repression may well be indirect.
Whether direct or indirect, the essential requirement of SXC/OGT for PcG repression provides an intriguing link to the well known but poorly reported dependence between the degree of PcG silencing and environmental conditions such as diet or temperature. The abundance of UDP-GlcNAc, the GlnNAc donor for OGT-dependent glycosylation, is clearly dependent on the availability of glucose. The requirement of glycosylation for effective PcG repression would therefore make this mechanism highly dependent on an energy-rich diet. This connection raises a wealth of exciting possibilities for the role of diet or calorie restriction on PcG repression and therefore on the wide range of processes that are affected by PcG mechanisms, including developmental processes, differentiation, cell cycle progression, X chromosome inactivation and tumor growth.
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