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Although the knowledge that nuclear and cytoplasmic proteins are modified with N-acetylglucosamine …

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- Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development

OGT, which modifies protein with O-GlcNAc, directly associateswith NCOAT, which reverses the modification with its O-GlcNAcasedomain, both in vivo and in vitro. NCOAT also has a HAT (Tolemanet al., 2004Go) domain putting it in the same complex as an HDAC,reversing at least part of the acetylation state of histonetails. This association of opposite enzymes is unusual in biology,because, on the surface, it would set up futile cycles for bothO-GlcNAcylation and histone acetylation. However, both enzymaticactivities can be modified in NCOAT (Toleman et al., 2004Go) invitro raising the potential that the OGT and O-GlcNAcase andthe HDAC and HAT activities are not on simultaneously. Furthermore,the ChIP assays reveal that on repressed DNA, there is detectablymore O-GlcNAc than on activated DNA. This observation impliesthat during repression, at least the OGT dominates the O-GlcNAcaseactivity, and during gene activation, this situation is reversed.Thus, in vivo, with natural levels as well as with overexpressionof the enzymes, there is no O-GlcNAc futile cycle.

Although, the targets of OGT were not addressed here, we haveshown previously that O-GlcNAc modification of at least oneof the activation domains of the ubiquitous transcription activator,Sp1, makes it incapable of activating transcription (Yang etal., 2001Go). However, other sites of O-GlcNAcylation on Sp1 couldactivate this transcription factor in different cells and ondifferent promoters (Goldberg et al., 2006Go). Also, the tailof RNA polymerase II itself is modified by OGT at phosphorylationsites, and the cycling of this enzyme may require O-GlcNAc asone of the modifications (Comer and Hart, 2001Go). Regardlessof whether GC boxes for Sp1 binding are present, basal transcriptionis repressed by OGT that is recruited to the promoter (Yanget al., 2002Go). Removal of this modification from Sp1, RNA polymeraseII, and perhaps other proteins (Jiang and Hart, 1997Go) by theO-GlcNAcase domain of NCOAT would thus be required to permita reversal of gene repression. Residing in the repression complexthrough association with OGT and other proteins, NCOAT is inthe correct place to reverse the repressive modification byOGT. Furthermore, NCOAT may stabilize the structure of the repressioncomplex. When combined with OGT, the two proteins bind the corepressormolecules better than OGT alone and this may apply to othercomplex associations as well. In addition, the presence of bothdomains in one protein, NCOAT, lends further credence to therole of O-GlcNAc in complementing HDAC-mediated gene repression.

For a promoter to transition from repression to activation requiresthe recruitment of signaling posttranslational modifiers (Bjorklundet al., 1999Go; Perissi et al., 2004Go), although the substratesof these modifiers have not yet been described. However, thederepressor NCOAT, by residing in corepression complexes, doesnot itself require recruitment like these other modifiers. Rather,NCOAT, which is strategically preplaced so that gene derepressioncan occur rapidly, must be posttranslationally modified so thatits enzymatic activities can be activated. When activation ofboth NCOAT enzymes cannot occur, gene transition from repressionto activation cannot occur in response to the hormone. In particular,NCOAT contains the only known O-GlcNAcase encoded in the genome.When it is replaced in the repression complex with a splicevariant of NCOAT putatively lacking only the O-GlcNAcase activity,there is a failure to remove the O-GlcNAc modification. In associationwith this failure to remove the O-GlcNAc modification on thegenes, an estrogen-responsive gene in breast ducts is not activatedduring puberty. Other genes in the mammary gland must be offas well, because the breast ducts do not develop properly intransgenic mice expressing this same splice variant. Althoughwild-type NCOAT is expressed at normal levels, it is ineffectiveat overcoming the dominant-negative NCOAT. This observationimplies that free NCOAT, not directed to the correct locationthrough OGT association, is ineffective in the system. Thisalso suggests that the removal of this O-GlcNAc modificationby NCOAT is required to allow this transition to activationby hormone-responsive genes.

The O-GlcNAcase of NCOAT has other functions. Relevant to geneactivation, its O-GlcNAcase activates the adenosine triphosphatase(ATPase) of the 19S cap of the proteasomes (Zhang et al., 2003Go).The active ATPases are required by the proteasomes-allowingcorepressors to be vectored to their destruction (Li et al.,2003Go). The local degradation by the proteasome of these repressorproteins, by decreasing their concentrations, could also contributeto gene activation. Although the NCOAT enzymes can be activated(Toleman et al., 2004Go), probably by posttranslational modification,the details of how they are activated remain unknown at thistime. The epitope masking of the initial state-specific NCOATantibody that can be relieved by phosphatases implies that atleast phosphorylation is one modification. We speculate thatthese phosphorylations of NCOAT may at least be part of thesignal that activates the enzymes of NCOAT. Whether phosphorylationscontrol the localization of NCOAT remains to be determined,but the antibody studies indicate that NCOAT’s associationwith OGT and other corepressors is not affected by the phosphorylationblocking the antibody. Nevertheless, the involvement of theO-GlcNAczyme, composed of OGT and NCOAT, in genes with estrogenreceptors imparts the necessary properties of reversibility,rapidity, and thoroughness required for homeostasis and suggeststhat the O-GlcNAc modification is more central to gene regulationthan simply providing a subtle nutritional input.

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